Baicalein-copper nanogel and preparation method and application thereof

Abstract

The application belongs to the technical field of nanometer material synthesis, and particularly relates to a baicalein-copper nanometer microgel as well as a preparation method and application thereof. The application forms nanometer wires by coordination of copper ions and baicalein, and then forms the nanometer microgel by coating with hyaluronic acid (HA). The method is carried out at normal temperature, and has the advantages of simple process, high yield and no toxic solvent. The obtained microgel has high stability (resistant to gastric acid), targeted delivery (CD44 receptor mediation) and multiple biological activities (antioxidation and anti-inflammation). Animal experiments show that the microgel can significantly relieve ulcerative colitis, has a low dose of 5 mg / kg and no toxicity, and provides a new strategy for the 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 (resistant 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 application provides the following technical solutions.

[0008] In a first aspect, the 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] Further preferably, the hydration radius of the nanowire core is 96 nm, and the hydration radius of the baicalein-copper nanogel is 110 nm.

[0013] Preferably, the baicalein-copper nanogel is stable in a simulated gastrointestinal fluid at pH 1.5 and pH 6.8 for ≥24 hours without dissociation or aggregation.

[0014] In a second aspect, the application provides a preparation method of the above 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.

[0015] 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.

[0016] Preferably, the mixed solvent is obtained by mixing water and an organic solvent selected from one or more of methanol, ethanol; the volume ratio of the water and the organic solvent is 1-5:1.

[0017] Preferably, the concentration of the adjusting agent NaOH solution is 0.5-1.0 M, and the pH is adjusted to 8-12.

[0018] Preferably, the stirring reaction is carried out at a temperature of 25-50℃ for 0.5-3 h.

[0019] Preferably, the baicalein solution is prepared by dissolving baicalein in an organic solution selected from one or more of methanol, ethanol, acetone, and the concentration of the baicalein solution is 10-60 mg / mL.

[0020] Preferably, the pH is adjusted to 10-12, the room temperature reaction is carried out for 12-24 h, and then the product is dispersed in deionized water by centrifugal washing to obtain a baicalein-copper nanowire dispersion; wherein the centrifugal speed is 8000-12000 rpm and the time is 10-15 min. Too low pH inhibits the coordination reaction, and too high pH causes copper ions to preferentially form copper hydroxide precipitate.

[0021] 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, the concentration of the baicalein-copper nanowire dispersion is 1-5 mg / mL; and the mixing reaction is carried out 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.

[0022] 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.

[0023] Preferably, the inflammatory diseases include ulcerative colitis, Crohn's disease, and periodontitis.

[0024] The beneficial effects achieved by the above one or more technical solutions of the application are as follows:

[0025] (1) The baicalein-copper nanomicrogel prepared by 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.

[0026] Specifically embodied in:

[0027] ①High structural stability: no aggregation or dissociation in simulated gastric fluid (pH 1.5) and intestinal fluid (pH 6.8) for ≥24 hours, i.e. resistant to gastric acid and trypsin degradation, ensuring the effectiveness of oral administration;

[0028] ②Low cytotoxicity (biocompatibility): when the concentration of copper ions is ≤100 μg / mL, the cell survival rate of L929 fibroblasts is >70%; when the concentration of copper ions is ≤20 μg / mL, the cell survival rate of RAW264.7 macrophages is ≈100%; and in animal experiments, no toxic reactions were observed in vivo at a dose as low as 5 mg / kg;

[0029] ③Strong antioxidant properties: significant ability to scavenge ABTS and DPPH free radicals; and can protect cells from oxidative stress damage;

[0030] ④Highly effective anti-inflammatory: 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 physical mixing; and in lipopolysaccharide-activated macrophages, the level of inflammatory factors is reduced by more than 50%.

[0031] ⑤Targeted delivery capability: mediated targeting by hyaluronic acid (HA), and the HA coating layer targets the inflammatory 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.

[0032] (2) The synthesis method of the present application has the characteristics of short process flow, green and safe raw materials, high yield, good stability, etc., 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.

[0033] (3) The nanowire structure prepared by the present application has good water dispersibility, high stability, uniform morphology and size, etc. 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 maintained to maintain structural stability; and the uniformity of the nanowire morphology is maintained to provide a high catalytic specific surface area.

[0034] (4) The baicalein-copper nanomicrogel synthesized by the present application has good biocompatibility, antioxidant properties, anti-inflammatory and antibacterial properties, and can exhibit good therapeutic effects in various chronic inflammatory diseases including inflammatory bowel disease, etc. In a DSS-induced mouse model, after 4 days of oral administration at a dose of 5 mg / kg, the colon length was 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

[0035] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated herein by reference. The embodiments of the present application, illustrated in the drawings and described below, serve to explain the present application.

[0036] Figure 1 Synthetic route of baicalein-copper nanowire structure prepared in Example 1 of the present application;

[0037] Figure 2 Transmission electron micrographs of baicalein-copper nanowire structure, baicalein-copper nanowire structure prepared in Example 1 of the present application, and baicalein-copper nanowire structure prepared in Example 6 of the present application;

[0038] Figure 3 Hydrated radius graph of baicalein-copper nanowire structure prepared in Example 1 of the present application;

[0039] Figure 4 Transmission electron micrographs of baicalein-copper nanowire structure prepared in Comparative Examples 1 to 2 of the present application;

[0040] Figure 5 Transmission electron micrographs 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;

[0041] Figure 6 Transmission electron micrographs of baicalein-copper nanocomposite prepared in Comparative Examples 5 to 7 of the present application;

[0042] Figure 7 X-ray photoelectron spectrograph of baicalein-copper nanowire structure prepared in Example 1 of the present application;

[0043] Figure 8 Stability test of baicalein-copper nanowire structure prepared in Example 1 of the present application in a simulated gastrointestinal fluid;

[0044] Figure 9 Peroxidase-like activity detection graph of baicalein-copper nanowire structure prepared in Example 1 of the present application at different concentrations;

[0045] Figure 10 ABTS scavenging graph of baicalein-copper nanowire structure prepared in Example 1 of the present application at different concentrations •+ UV spectrum;

[0046] Figure 11 DPPH scavenging UV spectrum of baicalein-copper nanowire structure prepared in Example 1 of the present application at different concentrations;

[0047] Figure 12Confocal images of intracellular reactive oxygen species detection and cell live / dead staining images of baicalein-copper nanogel, single copper salt, single baicalein and physical mixed group prepared for the present embodiment 1; wherein a) is the confocal image of intracellular reactive oxygen species detection, b) is the cell live / dead staining image;

[0048] Figure 13 Test image of baicalein-copper nanogel prepared for the present embodiment 1 regulating immune activity;

[0049] Figure 14 Test image of baicalein-copper nanogel prepared for the present embodiment 1 on L929 mouse fibroblast cell toxicity;

[0050] Figure 15 Test image of baicalein-copper nanogel prepared for the present embodiment 1 on RAW264.7 mouse macrophage cell toxicity;

[0051] Figure 16 Test image of baicalein-copper nanogel prepared for the present embodiment 1 on ulcerative colitis treatment effect;

[0052] Figure 17 Statistical image of colon length of ulcerative colitis treated by baicalein-copper nanogel prepared for the present embodiment 1. DETAILED DESCRIPTION

[0053] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the 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.

[0054] 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 embodiments.

[0055] Example 1 The present embodiment provides a baicalein-copper nanogel, and a preparation method thereof includes the following steps:

[0056] (1) Preparation of copper salt-containing suspension A

[0057] Compound (40 mg), PVP (80 mg) were weighed, and a mixed solution of 10 mL water and methanol (water: methanol = 5:1, volume ratio) was stirred and dissolved. The solution was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared light blue flocculation. After continuous stirring for 3 h, the solution became a uniform suspension A.

[0058] (2) Preparation of baicalein-copper nanowire

[0059] 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 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 aqueous dispersion C. The chemical reaction process is shown in Figure 1 , and the baicalein-copper nanowire structure obtained by transmission electron microscopy testing is shown in Figure 2 .

[0060] (3) Preparation of baicalein-copper nanogel

[0061] 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 until it was yellow-brown. The reaction was carried out at room temperature on a shaking table in the dark for 12 h. After the reaction was completed, the precipitate was collected by high-speed centrifugation (9000 rpm), and washed with ultrapure water. Finally, it was dispersed in ultrapure water to obtain the product. Transmission electron microscopy testing of the final structure found that the nanowire structure was still maintained (scale 100 nm), as shown in Figure 2 .

[0062] 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 showed that the hydration radius of the nanowire core (baicalein-copper nanowire structure) was 96 nm, and the hydration radius of the baicalein-copper nanogel was 110 nm. That is, compared with the baicalein-copper nanowire structure, the hydration radius of the nanowire increased after being coated with HA, which indirectly indicates that HA was successfully modified on the surface of the nanowire.

[0063] Example 2 The present embodiment provides a baicalein-copper nanogel, and a preparation method thereof includes the following steps:

[0064] The difference between the present 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:

[0065] (1) Preparation of copper salt-containing suspension A

[0066] Compound (50 mg), PVP (80 mg), 10 mL of a mixture of water and methanol (water: methanol = 5:1, volume ratio) was added 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, and after 3 h of continuous stirring, the solution became a uniform suspension A.

[0067] (2) Preparation of baicalein-copper nanowires

[0068] Baicalein (60 mg) was dissolved in 5 mL of methanol and ultrasonically dissolved 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, and redispersion with deionized water, baicalein-copper nanowire aqueous dispersion C was obtained.

[0069] (3) Preparation of baicalein-copper nanogel

[0070] 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, stirred and mixed, and the solution was yellow-brown. After reaction at room temperature on a shaking table for 12 h, the precipitate was collected by high-speed centrifugation (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water to obtain the product.

[0071] Example 3 The embodiment provides a baicalein-copper nanogel, and a preparation method thereof, which comprises the following steps:

[0072] The embodiment differs from embodiment 1 in that the mass ratio of baicalein to copper salt (CuSO4·5H2O) is 2:1, and the specific preparation steps are as follows:

[0073] (1) Preparation of copper salt-containing suspension A

[0074] Compound (30 mg), PVP (80 mg), 10 mL of a mixture of water and methanol (water: methanol = 5:1, volume ratio) was added 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, and after 3 h of continuous stirring, the solution became a uniform suspension A.

[0075] (2) Preparation of baicalein-copper nanowires

[0076] ​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.

[0077] (3) Preparation of baicalin-copper nanogels

[0078] 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.

[0079] Example 4 This embodiment provides a baicalin-copper nanogel, the preparation method of which includes the following steps:

[0080] 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:

[0081] (1) Preparation of suspension A containing copper salt

[0082] 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.

[0083] (2) Preparation of baicalin-copper nanowires

[0084] 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.

[0085] (3) Preparation of baicalin-copper nanogels

[0086] Take 5 mL baicalein-copper dispersion solution C (1 mg / mL) in a 25 mL open beaker, add 2 mL HA (2.5 mg / mL) aqueous solution, stir and mix evenly, the solution is yellow-brown, and place it in a shaking bed at room temperature for 12 h of reaction in the dark. 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 nanogel.

[0087] Example 5 The baicalein-copper nanogel provided in this embodiment has the following preparation method:

[0088] The difference between this embodiment and embodiment 1 is that the mass ratio of baicalein-copper nanowires to hyaluronic acid (HA) is 2.5:1, and the specific preparation steps are as follows:

[0089] (1) Preparation of copper salt-containing suspension A

[0090] Take the compound (40 mg), PVP (80 mg), add 10 mL of a mixture of water and methanol (water:methanol = 5:1, volume ratio), stir and dissolve, and the solution is light blue. Adjust the pH to 10 using NaOH (1 M), and the solution gradually appears as light blue flocculation. After stirring for 3 h, the solution becomes a uniform suspension A.

[0091] (2) Preparation of baicalein-copper nanowires

[0092] Dissolve baicalein (60 mg) in 5 mL of methanol to obtain solution B, which is light yellow-green. Then add it dropwise to solution A, and the color gradually changes from light blue to tan. After stirring for half an hour, adjust the pH to 12 using NaOH (1 M), and the solution gradually becomes clear. After stirring for 12 h, centrifuge and wash, and then redisperse in deionized water to obtain a baicalein-copper nanowire aqueous dispersion solution C.

[0093] (3) Preparation of baicalein-copper nanogel

[0094] Take 5 mL baicalein-copper dispersion solution C (1 mg / mL) in a 25 mL open beaker, add 2 mL HA (2.5 mg / mL) aqueous solution, stir and mix evenly, the solution is yellow-brown, and place it in a shaking bed at room temperature for 12 h of reaction in the dark. 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 nanogel.

[0095] Example 6 The baicalein-copper nanogel provided in this embodiment has the following preparation method:

[0096] (1) Preparation of the suspension A containing copper salt

[0097] The compounds were weighed (40 mg), PVA (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). The solution gradually appeared light blue flocculation. After 3 h of continuous stirring, the solution became a uniform suspension A.

[0098] (2) Preparation of baicalein-copper nanowires

[0099] 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. 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). 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.

[0100] (3) Preparation of baicalein-copper nanogel

[0101] 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 stirred to mix. The solution was brown. It was placed in a shaking incubator 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, it was dispersed in ultrapure water. Transmission electron microscopy of the final structure found that it was a mesh structure of intertwined nanowires (scale 100 nm), as shown in FIG. 1c. Figure 2

[0102] Comparative Example 1 This comparative example provides a baicalein-copper nanocomposite, and the preparation method thereof comprises the following steps:

[0103] The difference between this comparative example and Example 1 is that the mass ratio of baicalein to copper salt (CuSO4·5H2O) is 2.125:1. The specific preparation steps are as follows:

[0104] (1) Preparation of the suspension A containing copper salt

[0105] The compounds were weighed ​​(40 mg), PVP (80 mg), 10 mL of a mixture of water and methanol (water: methanol = 5:1, volume ratio) was added 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, and after 3 h of continuous stirring, the solution became a uniform suspension A.

[0106] (2) Preparation of baicalein-copper nanowires

[0107] Baicalein (85 mg) was dissolved in 5 mL of methanol and ultrasonically dissolved to obtain solution B, which was yellow-green. Then it was added dropwise to solution A, and the color changed from light blue to tan quickly, and after stirring for half an hour, NaOH (1 M) was added to adjust the pH to 12, and the solution appeared dark brown and flocculated. After stirring for 12 h, centrifugation and washing, and redispersion with deionized water, a baicalein-copper nanostructure aqueous dispersion C was obtained, and the baicalein-copper nanostructure obtained is shown in FIG. 1a (scale 100 nm). It can be seen that the structure is not a nanowire structure, but a network crosslinking material. Figure 4

[0108] (3) Preparation of baicalein-copper nanocomposites

[0109] 5 mL of baicalein-copper nanostructure aqueous 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 stirred to mix, and the solution was yellow-brown. After 12 h of reaction at room temperature on a shaking table in the dark, the precipitate was collected by high-speed centrifugation (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water to obtain a baicalein-copper nanocomposite.

[0110] Comparative Example 2 This comparative example provides a baicalein-copper nanocomposite, and the preparation method thereof comprises the following steps:

[0111] 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:

[0112] (1) Preparation of copper salt-containing suspension A

[0113] Compound (40 mg), PVP (80 mg), 10 mL of a mixture of water and methanol (water: methanol = 5:1, volume ratio) was added 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, and after 3 h of continuous stirring, the solution became a uniform suspension A.

[0114] (2) Preparation of baicalein-copper nanowires​​

[0115] 20 mg of baicalein was dissolved in 5 mL of methanol and sonicated to obtain solution B, which was pale yellow. This solution was then added dropwise to solution A, and the color rapidly changed from pale blue to light brown. 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 an aqueous dispersion C of baicalein-copper nanostructures. The obtained baicalein-copper nanostructures are shown below. Figure 4 As shown in b (scale bar 100 nm), it can be seen that the structure does not exhibit a nanowire structure, but rather a sheet or block structure.

[0116] (3) Preparation of baicalin-copper nanocomposite

[0117] 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.

[0118] Comparative Example 3 This comparative example provides a baicalin-copper nanocomposite, the preparation method of which includes the following steps:

[0119] The difference between this comparative example and Example 1 is that the mass ratio of baicalin-copper nanowires to hyaluronic acid (HA) is 5:1. The specific preparation steps are as follows:

[0120] (1) Preparation of suspension A containing copper salt

[0121] 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.

[0122] (2) Preparation of baicalin-copper nanowires

[0123] 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.

[0124] (3) Preparation of baicalein-copper nanocomposite

[0125] 5 mL of baicalein-copper dispersion solution 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 until it turned reddish brown. It was placed in a shaking bed at room temperature and avoided light for 12 h. After the reaction was completed, the precipitate was collected by high-speed centrifuge (9000 rpm), washed with ultrapure water, and finally dispersed in ultrapure water. The final structure was tested and found that uniform nanowires were not formed, but particles and nanowires coexisted (scale 20 nm), as shown in Figure 5 a.

[0126] Comparative Example 4 This comparative example provides a baicalein-copper nanocomposite, and the preparation method thereof comprises the following steps:

[0127] 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:

[0128] (1) Preparation of copper salt-containing suspension A

[0129] Compound (40 mg) and PVP (80 mg) were weighed, and 10 mL of a mixture of water and methanol (water:methanol = 5:1, by volume) was added and stirred to dissolve the solution, which turned light blue. The pH was adjusted to 10 using NaOH (1 M), and light blue floc gradually appeared in the solution. After continuous stirring for 3 h, the solution became a uniform suspension A.

[0130] (2) Preparation of baicalein-copper nanowires

[0131] Baicalein (60 mg) was dissolved in 5 mL of methanol and ultrasonically dissolved 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, the solution was centrifuged and washed with deionized water, and a baicalein-copper nanowire aqueous dispersion solution C was obtained.

[0132] (3) Preparation of baicalein-copper nanocomposite

[0133] The 5 mL baicalein-copper dispersion solution C (1 mg / mL) was placed in a 25 mL open beaker, 1 mL HA (20 mg / mL) aqueous solution was added, and the solution was stirred and mixed until it became a yellow-brown jelly. The solution was placed in a shaking table at room temperature and reacted for 12 h in the dark. Finally, the solution was washed and dispersed again by ultrasonic treatment. The structure was as shown in FIG. 1C. Figure 5 The structure was as shown in FIG. 1C.

[0134] Comparative Example 5 This comparative example provided a baicalein-copper nanocomposite, and the preparation method included the following steps:

[0135] The difference between this comparative example and Example 1 was that NaOH was not added to adjust the pH of the reaction solution.

[0136] The compound (40 mg) and PVP (80 mg) were weighed, and a mixture of 10 mL water and methanol (water:methanol = 5:1, by volume) was added and stirred to dissolve the solution, which was light blue. Then baicalein (60 mg) was dissolved in 5 mL methanol and ultrasonically dissolved, and then added dropwise to the copper salt solution described above. After stirring for 12 h, the solution was centrifuged, washed, and redispersed with deionized water.

[0137] The 5 mL dispersion solution was placed in a 25 mL open beaker, 1 mL HA (2.5 mg / mL) aqueous solution was added, and the solution was stirred and mixed until it became a yellow-brown jelly. 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 solution was washed and dispersed again by ultrasonic treatment. The morphology was irregular flake, and uniform nanowire structure was not formed (scale 50 nm), as shown in FIG. 1A. Figure 6 The structure was as shown in FIG. 1C.

[0138] Comparative Example 6 This comparative example provided a baicalein-copper nanocomposite, and the preparation method included the following steps:

[0139] The difference between this comparative example and Example 1 was that in step (2), the pH was adjusted to 8.

[0140] (1) Preparation of copper salt-containing suspension A

[0141] The compound (40 mg) and PVP (80 mg) were weighed, and a mixture of 10 mL water and methanol (water:methanol = 5:1, by volume) was added and stirred to dissolve the solution, which was light blue. The pH was adjusted to 10 using NaOH (1 M), and the solution gradually appeared as a light blue flocculent material. After continuous stirring for 3 h, the solution became a uniform suspension A.

[0142] (2) Preparation of baicalein-copper nanowires

[0143] 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 8. After stirring for 12 h, centrifugal washing was performed, and redispersion was performed using deionized water to obtain a baicalein-copper water dispersion solution C.

[0144] (3) Preparation of a baicalein-copper nanocomposite

[0145] 5 mL of baicalein-copper dispersion solution 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), and washed with ultrapure water, and finally dispersed in ultrapure water. The final structure is shown in Figure 6 b (scale 50 nm). It can be seen that no nanowires are formed in the final product, but only extremely small and poorly crystalline nanoparticles.

[0146] Comparative Example 7 This comparative example provides a baicalein-copper nanocomposite, and the preparation method thereof comprises the following steps:

[0147] The difference between this comparative example and Example 1 is that in step (2), the pH is adjusted to 14.

[0148] (1) Preparation of a copper salt-containing suspension A

[0149] Compound (40 mg), PVP (80 mg) was weighed, and a mixture of 10 mL of water and methanol (water:methanol = 5:1, by volume) was stirred and dissolved to obtain a light blue solution. NaOH (1 M) was used to adjust the pH to 10, and light blue flocculation appeared in the solution. After continuous stirring for 3 h, the solution became a uniform suspension A.

[0150] (2) Preparation of a baicalein-copper nanowire

[0151] 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 14. After stirring for 12 h, centrifugal washing was performed, and redispersion was performed using deionized water to obtain a baicalein-copper water dispersion solution C.

[0152] (3) Preparation of a baicalein-copper nanocomposite

[0153] 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 at 9000 rpm and washed with ultrapure water. The precipitate was then dispersed in ultrapure water, and the final structure was as shown in the figure. Figure 6 As shown in c (scale bar 50 nm), it can be seen that a large number of random aggregate structures are present.

[0154] Comparative Example 8 This comparative example provides a baicalin-copper nanocomposite, the preparation method of which includes the following steps:

[0155] Weigh the compound (40 mg) was 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, baicalein (60 mg) was dissolved in 5 mL of methanol and sonicated until dissolved. This solution was then added dropwise to the aforementioned copper salt solution. After stirring and reacting for 12 h, the solution was centrifuged, washed, and redispersed with deionized water to obtain a physical mixture of baicalein and copper salt.

[0156] Experimental Example 1 This experiment characterizes the structure and properties of the baicalin-copper nanogel prepared in Example 1.

[0157] like Figure 7 As shown in Figure a, the synthesized baicalin-copper nanogel contains four elements: Cu, O, N, and C. Figure 7 As shown in Figure b, after baicalin coordinates with metallic copper, the electron cloud density redistributes. The fine spectrum of the copper ions shows peaks at 932.8 / 953.8 eV and 931.2 / 951.4 eV, corresponding to Cu, respectively. 2+ and Cu + (Mixed valence state copper) proves that baicalin successfully coordinates with copper ions, and that the copper ions contained in the nanowires are in the Cu valence state. 2+ and Cu + The mixed valence state is beneficial for its subsequent antioxidant properties in neutralizing free radicals such as reactive oxygen species and nitrogen.

[0158] like 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 is added to 4 mL of the simulated gastric solution (pH = 1.5) containing pepsin (3.2 mg / mL), and after 24 h, the supernatant is collected by centrifugation at 9000 r / min for 10 min. Similarly, another 200 μL of the nanogel solution is mixed with 4 mL of the simulated intestinal solution (pH = 6.8) containing trypsin (10 mg / mL), and after 24 h, the supernatant is collected by centrifugation at 9000 r / min for 10 min. Then, the gastrointestinal stability of the baicalein-copper nanogel is evaluated by transmission electron microscopy (TEM).

[0159] Application Example 1 The 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.

[0160] (1) Peroxidase-like enzyme removal experiment

[0161] ① Test process: The prepared samples are dispersed in ultrapure water to prepare a solution with a concentration of 0.2 μg / mL, and then 50 μL of H2O2 (50 mM) and 100 μL of 3,3',5,5'-tetramethylbenzidine solution TMB (10 mg / mL) are added to 2 mL of the above solution at room temperature. After 5 min of reaction, the absorbance value at 652 nm is recorded using a visible-ultraviolet spectrophotometer. The specific values are shown in Tables 1-2:

[0162] Table 1

[0163]

[0164] Table 2

[0165]

[0166] 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 removal effect; compared with Examples 2-6, the baicalein-copper nanogel prepared in Example 1 has the optimal peroxidase-like enzyme removal effect.

[0167] ② Test procedure: The baicalin-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, at room temperature, H2O2 (50 mM) and 100 µL of 3,3',5,5'-tetramethylbenzidine solution (10 mg / mL) were added to 2 mL of the above-prepared sample solutions, respectively. After reacting for 5 min, the absorbance value at 652 nm was recorded using a visible-ultraviolet spectrophotometer. The test results are shown in Table 3.

[0168] Table 3

[0169]

[0170] From Table 3 and Figure 9 It can be seen that the absorbance value of the baicalin-copper nanogel prepared in Example 1 of the present invention gradually decreases with the increase of sample solution concentration, and the peroxidase-like scavenging effect is enhanced accordingly.

[0171] (2) ABTS •+ Free radical scavenging experiment and DPPH • Free radical scavenging experiment

[0172] ①ABTS• + Free radical scavenging experiment:

[0173] 1) Prepare ABTS by mixing 1 mL of potassium persulfate solution (37.84 mg / mL, 2.6 mM) with 5 mL of ABTS solution (3.84 mg / mL, 7.4 mM) in the dark for 12 h. •+ The working solution was prepared for use. Subsequently, the ABTS stock solution was diluted with 80% (v / v) methanol, water, or PBS to obtain the working solution, adjusting its absorbance to between 0.6 and 1.0. Then, the sample solutions prepared in the examples and comparative examples (0.2 μg / mL) were added respectively, and the reactions were carried out in the dark for 30 min. The absorbance of each group of samples at 734 nm was measured, and the ABTS values ​​were calculated. •+ Free radical scavenging rate, the test data are shown in Tables 4 and 5:

[0174] Table 4

[0175]

[0176] Table 5

[0177]

[0178] As shown in Tables 4-5, compared with the comparative examples, the baicalin-copper nanogels prepared in Examples 1-6 of this invention have 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.

[0179] 2) 1 mL of potassium persulfate solution (37.84 mg / mL, 2.6 mM) and 5 mL of ABTS solution (3.84 mg / mL, 7.4 mM) were placed in a dark environment for 12 h 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, and the absorbance thereof was between 0.6 and 1.0. Then, 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.

[0180] Table 6

[0181]

[0182] It can be seen from Table 6 and Figure 10 the ABTS radical scavenging rate of the baicalein-copper nanogel prepared in example 1 gradually increases with the increase of the concentration of the sample solution, and the scavenging effect is enhanced. •+

[0183] ②DPPH • radical scavenging experiment

[0184] 1) DPPH (11.8 mg / 2mL) powder was dissolved in ethanol to obtain a DPPH working solution having 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, and the DPPH scavenging rate was calculated. The test data are shown in Tables 7~8.

[0185] Table 7

[0186]

[0187] Table 8

[0188]

[0189] ​As shown in Tables 7-8, compared with the comparative examples, the baicalein-copper nanometer microgel prepared in the inventive examples 1-6 has stronger DPPH free radical scavenging effect; compared with the examples 2-6, the baicalein-copper nanometer microgel prepared in the inventive example 1 has the optimal DPPH free radical scavenging effect.

[0190] 2) Dissolve DPPH (11.8 mg / 2mL) powder with ethanol to obtain DPPH working solution having an absorption peak at 517 nm, and note that the present working solution is prepared on the spot. Add different concentrations of baicalein-copper nanometer microgel (0.2, 0.4, 1.0 μg / mL) solution to the DPPH working solution, and detect the absorbance value at 517 nm after the reaction in the dark for 30 min, and calculate the DPPH scavenging rate, and the test data are shown in Table 9:

[0191] Table 9

[0192]

[0193] As shown in Tables 9 and Figure 11 , the DPPH free radical scavenging rate of the baicalein-copper nanometer microgel prepared in the inventive example 1 gradually increases with the increase of the sample solution concentration, and the scavenging effect is enhanced accordingly.

[0194] In summary: the absorbance of example 1 is the lowest (0.117), and the free radical scavenging capacity is the strongest; while in the comparative example 1, the mass ratio of baicalein to copper salt is 2.125:1, and a reticular crosslinked material is formed, which is not a nanometer wire structure and loses the catalytic site; in the comparative example 2, the mass ratio of baicalein to copper salt is 0.5:1, and a flaky or massive structure is formed, the size is not uniform, the active site is not exposed enough, and the peroxidase-like scavenging effect and the free radical scavenging effect are both poor;

[0195] In the comparative example 3, 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 the comparative example 4, 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 sites in the core; in the comparative example 5 (random flaky without NaOH regulation), the activity is significantly reduced, and both the peroxidase-like scavenging effect and the free radical scavenging effect are poor;

[0196] In the 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 alkali will destroy the coordination bond, resulting in agglomeration, and both the peroxidase-like scavenging effect and the free radical scavenging effect are poor;

[0197] In addition, since the coordination of baicalein and copper enhances the electron transfer ability and synergistically improves the enzyme-like activity, the peroxidase-like scavenging effect and the free radical scavenging effect of the embodiment are much higher than those of single copper salt, single baicalein and physical mixture; and a higher concentration of sample solution can provide more active sites, accelerate the free radical scavenging kinetics, and thus improve the scavenging effect.

[0198] Application Example 2 The application example provides an exploration of the ROS scavenging ability of the sample prepared in the embodiment and the comparative example and the immunomodulatory activity on M1 macrophages

[0199] ①ROS in cells is considered to be a key messenger that amplifies inflammatory response and regulates the generation of inflammatory mediators. In order to evaluate whether the baicalein-copper nanowire microgel prepared in the embodiment 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, 2', 7'-dichlorofluorescein diacetate (DCFH-DA) probe was used to visualize the ROS signal in cells, and Calcein AM / PI cell live and dead double staining kit was used to detect the live and dead situation of cells after oxidative stress. In the ROS detection, the stronger the green fluorescence, the higher the level of active oxygen in the 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 the comparative example 8 and single lipopolysaccharide) is the same as that of the embodiment.

[0200] The test results are shown in a~b in Figure 12 Fig. a is a confocal image of intracellular active oxygen detection, from which it can be seen that, compared with untreated cells, the green fluorescence signal of the hydrogen peroxide stimulation group is significantly enhanced, and the baicalein-copper nanowire microgel prepared in the embodiment 1 has higher ROS scavenging capacity compared with baicalein, copper ions and physical mixture of baicalein and copper ions at the same concentration. In addition, it can also be seen from the cell live and dead staining image of Fig. b that, based on its good ROS scavenging activity, the baicalein-copper nanowire microgel prepared in the embodiment 1 can effectively protect cells from apoptosis, and this group also shows the most number of live cells (the strongest green fluorescence).

[0201] ②The imbalance of inflammatory mediators seriously damages the intestinal barrier function of UC patients and triggers excessive immune response. 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 influence of baicalein-copper nanowire microgel on the secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 by M1 macrophages was determined by enzyme-linked immunosorbent assay (ELISA).

[0202] As shown in Figure 13 , the levels of TNF-α, IL-1β and IL-6 in RAW264.7 cells treated with different treatments and activated with 0.2 μg / mL of LPS for 24 h were tested. As can be seen from the figure: compared with normal RAW264.7 cells, the expression of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α) 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 of the present application had the best inhibitory effect on the expression of pro-inflammatory cytokines among all the treatment groups, showing stronger anti-inflammatory effect.

[0203] Application Example 3 : The present application example provides safety and effectiveness determination of the baicalein-copper nanowire microgel prepared in example 1 for ulcerative colitis

[0204] ① Cytotoxicity test experiment:

[0205] 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 microgel (copper ion concentrations were 3.175, 6.25, 12.5, 25, 50, 100 μg / mL) were added, and then the cells were cultured for another 24 h for toxicity detection.

[0206] The test results are shown in Figure 14 , with the increase of copper ion concentration, the cell survival rate was maintained above 70%, thus indicating that the baicalein-copper nanowire microgel prepared in the present example 1 has good biocompatibility, and no obvious toxicity to mouse fibroblasts.

[0207] 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 microgel (copper ion concentrations were 1, 2.5, 5, 10, 20 μg / mL) were added, and then the cells were cultured for another 24 h for toxicity detection.

[0208] The test results are shown in Figure 15 , with the increase of copper ion concentration, the cell survival rate was maintained above and below 100%, thus indicating that the baicalein-copper nanowire microgel prepared in the present example 1 has good biocompatibility, and no obvious toxicity to mouse macrophages.

[0209] 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.

[0210] 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).

[0211] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. 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 baicalein-copper nanomicrogel, characterized in that, The baicalein-copper nanomicrogel 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. The pH of the coordination reaction is controlled to be 10-12.

2. The baicalein-copper nanogel of claim 1, wherein, The copper in the nanowire core is in mixed valence Cu + / Cu 2+ The nanowire core is formed by coordination of copper ions with baicalein.

3. The baicalein-copper nanogel of claim 1, wherein the copper is present in an amount of 0.1 to 10 wt%. The hydration radius of the nanowire core is 90-98 nm, and the hydration radius of the baicalein-copper nanomicrogel is 100-120 nm.

4. The baicalein-copper nanogel of claim 3, wherein, The hydration radius of the nanowire core is 96 nm, and the hydration radius of the baicalein-copper nanomicrogel is 110 nm.

5. The baicalein-copper nanogel of claim 1, wherein, The baicalein-copper nanomicrogel is stable in a simulated gastrointestinal fluid at pH 1.5 and pH 6.8 for ≥24 hours without dissociation or aggregation.

6. A method for preparing baicalein-copper nanogel according to any one of claims 1-5, characterized in that, The method 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 nanomicrogel. The concentration of the NaOH solution as the regulator is 0.5-1.0 M, and the pH is adjusted to 10-12.

7. The production method according to claim 6, wherein The copper salt is selected from one of CuSO4·5H2O and CuSO4; the surfactant is selected from one or more of polyvinylpyrrolidone and polyvinyl alcohol; and the mass ratio of the copper salt to the surfactant is 0.2-1:

1.

8. The production method according to claim 6, wherein The mixed solvent is obtained by mixing water and an organic solvent, and 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.

9. The production method according to claim 6, wherein The stirring reaction is performed at a temperature of 25-50°C for 0.5-3 h.

10. The production method according to claim 6, wherein The baicalein solution is prepared by dissolving baicalein in an organic solution, 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.

11. The production method according to claim 6, wherein The pH is adjusted to 10-12, the room temperature reaction is performed for 12-24 h, then the product is centrifuged and washed, and the product is dispersed in deionized water to obtain the baicalein-copper nanowire dispersion; the centrifugation is performed at a speed of 8000-12000 rpm for 10-15 min.

12. The production method according to claim 6, wherein The volume ratio of the baicalein-copper nanowire dispersion to the hyaluronic acid aqueous solution is 5-10:1; the concentration of the hyaluronic acid aqueous solution is 2.5-10 mg / mL, 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°C for 12-24 h.

13. The production method according to claim 12, wherein The volume ratio of the baicalein-copper nanowire dispersion to the hyaluronic acid aqueous solution is 5:

1.

14. Use of the baicalein-copper nanomicrogel according to any one of claims 1-5 in the preparation of a drug for treating ulcerative colitis.

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