Copper ion-luteolin nano-composite and application thereof in treatment of inflammatory bowel disease

Abstract

The invention discloses a copper ion-luteolin nano-composite and application thereof in treatment of inflammatory bowel disease, the obtained nano-composite has very strong stability and active oxygen scavenging ability, and has excellent anti-oxidation and anti-inflammatory ability, specifically, the nano-composite is negatively charged, can effectively target intestinal inflammation lesion, and can be used for treating inflammatory bowel disease, so that the copper ion-luteolin nano-composite can be applied to treatment of inflammatory bowel disease. Mucosal barrier repair is promoted, intestinal flora is regulated, and the inflammation microenvironment is remarkably regulated; the antioxidant and anti-inflammatory capacities of cells can be enhanced by regulating and controlling an oxidative stress pathway of a nuclear factor erythroid 2 related factor 2 / heme oxygenase-1 (Nrf2 / HO-1) and a nuclear factor kappa B (NF-kappa B) signal pathway; the compound shows a good treatment effect in a typical inflammatory bowel disease mouse model, including ulcerative colitis and Crohn's disease. The invention provides a new way for constructing a multi-layer treatment platform for treating the inflammatory bowel disease by using natural products.

Description

Technical Field

[0001] The invention belongs to the technical field of nano material preparation, and specifically relates to a copper ion-luteolin nanocomposite and a preparation method thereof and application thereof in the treatment of inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory disease with a rapidly increasing incidence worldwide, especially in newly industrialized countries. The pathogenesis of IBD is associated with oxidative stress, inflammatory response, disruption of intestinal barrier function, imbalance of intestinal microbiota, and dysregulation of the mucosal immune system. Traditional medical interventions for IBD often fail to address its root causes, and the longer the inflammation persists, the higher the risk of intestinal and systemic complications. Therefore, developing rapid and effective strategies is key to treating IBD.

[0003] During the development of inflammatory bowel disease, complex molecular interactions and cellular communications occur between different proinflammatory cytokines, chemokines, inflammatory cells, and immune cells, such as: (1) Oxidative stress and cell damage: There is an excess of reactive oxygen species above normal levels in the inflammatory microenvironment. Intestinal epithelial cell damage is caused by an imbalance of oxidative stress and a decrease in antioxidant capacity caused by high concentrations of reactive oxygen species. This imbalance oxidizes proteins, lipids, and damages DNA molecules. (2) Intestinal barrier destruction and inflammation: The loss of intestinal epithelial cells destroys the mechanical barrier of the intestine, and the entry of neutrophils and other pathogens aggravates the damage to colon tissue. On the other hand, excessive reactive oxygen species can affect macrophage differentiation and activate inflammatory factors to cause inflammation. M2 macrophages play a role in immunosuppression and tissue repair, and usually produce very few anti-inflammatory cytokines. This leads to chronic inflammation and triggers a series of inflammatory cascade reactions. (3) Microbial imbalance: There is increasing evidence that intestinal inflammation and permeability problems caused by microbial imbalance play an important role in the progression of inflammatory bowel disease. Restoring intestinal homeostasis is a complex challenge that cannot be achieved by a single treatment. Therefore, there is an urgent need to develop a series of multidimensional regulatory strategies that can both alleviate the symptoms of the intestinal inflammatory microenvironment and promote better long-term management outcomes, thereby ensuring the safe and effective treatment of inflammatory bowel disease.

[0004] Natural active small molecules have the advantages of few side effects, reasonable price and easy access, and they hold great promise in the treatment of inflammatory bowel disease. Luteolin is a flavonoid with unique antioxidant properties, which has attracted extensive attention due to its multiple biological activities, including anti-inflammatory, anticancer and cardiovascular protection. Luteolin exhibits significant efficacy in regulating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. It works by upregulating antioxidant enzyme genes such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1), thereby alleviating oxidative stress, and inhibiting inflammation by targeting proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Therefore, the regulation of Nrf2 by luteolin is considered to be an effective strategy for the treatment of colitis. However, the application of natural active small molecules is often limited by challenges such as instability, lack of targeting and relatively low bioavailability. In addition, luteolin is susceptible to oxidative degradation due to its highly unsaturated structure, which limits its solubility and bioavailability in water. Summary of the invention

[0005] Based on the shortcomings of the above-mentioned prior art, the present invention provides a copper ion-luteolin nanocomposite and its preparation method and application in the treatment of inflammatory bowel disease, aiming to improve the low bioavailability of natural active small molecules by using the strategy of metal ion nano-size. Specifically, copper is an essential trace element for the human body. When copper is oxidized (Cu 2+ ) and reduction (Cu + ) forms, both forms have inherent redox properties, which are essential for maintaining redox homeostasis. Therefore, the present invention utilizes the complexation of copper ions with luteolin to enhance the enzymatic catalytic performance and antioxidant activity of luteolin, thereby improving the overall active oxygen scavenging efficiency and bioavailability.

[0006] In order to solve the technical problem, the present invention adopts the following technical solution:

[0007] The present invention first provides a method for preparing a copper ion-luteolin nanocomposite, which is characterized in that anhydrous CuSO4 and luteolin solution are uniformly dispersed in water, mixed and stirred in the presence of polyvinylpyrrolidone (PVP), and the copper ion-luteolin nanocomposite is obtained. Specifically, the method comprises the following steps:

[0008] First, PVP was dissolved in deionized water, and then anhydrous CuSO4 powder was added and stirred until clear. Then, luteolin was added and stirred at room temperature for 3 to 4 days, and then centrifuged to remove excess luteolin. The resulting supernatant was ultrafiltered to remove excess PVP and copper ions, thereby obtaining a dispersion of copper ion-luteolin nanocomplex.

[0009] Furthermore, the mass ratio of luteolin, anhydrous CuSO4 and PVP is 1:5-6:25-35.

[0010] Furthermore, the centrifugal speed is 8000-12000 rpm and the centrifugal time is 8-12 min.

[0011] Furthermore, the ultrafiltration uses a 100 kDa ultrafiltration tube.

[0012] The copper ion-luteolin nanocomposite prepared by the present invention can be used to prepare drugs for preventing and / or treating inflammatory bowel disease. The principle is that based on the high stability of the copper ion-luteolin nanocomposite, the negatively charged copper ion-luteolin nanocomposite can effectively target positively charged inflammatory lesions in the intestine, reduce reactive oxygen species in the inflammatory site, and protect intestinal epithelial cells. While promoting the polarization of macrophages to the M2 phenotype, it also promotes the nuclear translocation of Nrf2, thereby activating the expression of downstream antioxidant enzyme HO-1 to combat oxidative stress damage. The copper ion-luteolin nanocomposite inhibits the nuclear factor κB (NF-κB) signaling pathway by downregulating phosphorylated i-κB kinase β (p-IKKβ) and phosphorylated NF-κB P65 (p-P65), thereby reducing proinflammatory factors and effectively regulating oxidative stress imbalance and excessive inflammation. In addition, the copper ion-luteolin nanocomposite restores the level of tight junction-related proteins, improves intestinal barrier function, and also enhances the abundance and diversity of intestinal microbiota. Achieve precise, effective and multi-pronged treatment of inflammatory bowel disease.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. The present invention synthesizes a copper ion-luteolin nanocomposite in an aqueous solution. The synthetic raw materials used are simple, the method is simple, the reaction conditions are mild, the obtained product has uniform morphology, the production cost is low, and it is suitable for industrial scale-up production.

[0015] 2. The copper ion-luteolin nanocomplex prepared by the present invention can target the inflammatory sites in the intestine and interrupt the inflammatory circuit of inflammatory bowel disease, effectively prevent the onset of inflammatory bowel disease, and achieve accurate, effective and long-term treatment of inflammatory bowel disease.

[0016] 3. The copper ion-luteolin nanocomplex prepared by the present invention is expected to become a composite nanomaterial that coordinates the multi-faceted regulation of the inflammatory microenvironment by regulating the oxidative stress pathway Nrf2 and the immune pathway NF-κB and regulating the disorder of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a transmission electron microscope (TEM) image of the copper ion-luteolin nanocomposite prepared in Example 1 ( Figure 1 (a)) and Zeta potential diagram ( Figure 1 (b) in the figure.

[0018] Figure 2 The antioxidant performance test diagram of the copper ion-luteolin nanocomposite prepared in Example 1: (a) ABTS· + scavenging and DPPH·performance, (b)·OH scavenging activity, H2O2 scavenging activity and·O2 - Scavenging activity (SOD-like and CAT-like enzyme activities).

[0019] Figure 3 The copper ion-luteolin nanocomplex prepared in Example 1 scavenges ROS and protects cells. Figure: (a) Cell live and dead fluorescence staining, (b) DCFH-DA staining of ROS in cells.

[0020] Figure 4 The copper ion-luteolin nanocomplex prepared in Example 1 regulates immune activity test diagram: the level of inflammatory factors.

[0021] Figure 5 The effect of the copper ion-luteolin nanocomplex on Nrf2 in Example 1; Nrf2 fluorescence staining.

[0022] Figure 6 Figure 5 shows the colon length of each group in Example 5 for the prevention of DSS-induced inflammatory bowel disease in mice: (a) mouse colon, (b) statistics of mouse colon length

[0023] Figure 7 Figure 5 shows the H&E staining of colon sections of each group in Example 5 for the prevention of DSS-induced inflammatory bowel disease in mice: (a) H&E staining of mouse colon sections, (b) H&E pathological scoring statistics of mouse colon

[0024] Figure 8 The preventive effect of each group in Example 5 on DSS-induced inflammatory bowel disease in mice is shown in the figure: (a) mouse body weight, (b) disease activity index;

[0025] Fig. 9 The therapeutic effects of each group in Example 6 on DSS-induced inflammatory bowel disease in mice are shown in the following figures: (a) mouse colon, (b) mouse colon length statistics, (c) mouse body weight, (d) disease activity index, and (e) immunohistochemical staining.

[0026] Fig.10 The figures show the intestinal flora regulation effect of each group in Example 6 on DSS-induced inflammatory bowel disease in mice: (a) relative abundance of the top 20 intestinal microbial phylum-level classifications, (b) α diversity, and (cf) relative abundance of selected taxa in the family-level classifications. DETAILED DESCRIPTION

[0027] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0028] Example 1

[0029] In this example, the copper ion-luteolin nanocomposite was prepared according to the following steps:

[0030] First, 1200 mg of PVP was dissolved in 40 mL of deionized water. Then, 210 mg of anhydrous CuSO4 powder was added to the solution, stirred until clear, and then 40 mg of luteolin was added, stirred at room temperature for 72 hours, and centrifuged at 10000 rpm for 10 min to remove excess luteolin. The resulting supernatant was then filtered with a 100KD ultrafiltration tube to remove excess PVP and copper ions, thereby obtaining a dispersion of copper ion-luteolin nanocomplexes (denoted as CuL, quantitatively measured by freeze-drying, the concentration of the nanocomplex in the dispersion obtained in this embodiment was 40 mg / mL).

[0031] like Figure 1 As shown in the TEM image of (a), the copper ion-luteolin nanocomposite obtained in this example is uniform nanoparticles. Figure 1 As shown in the Zeta potential diagram of (b), the detection potential was -35.7±3.83 mV, indicating that the copper ion-luteolin nanocomplex was successfully synthesized and carried a negative charge, which could target the positively charged inflamed colon.

[0032] Example 2

[0033] This example uses a free radical scavenging experiment to verify the antioxidant properties of the copper ion-luteolin nanocomposite (CuL) prepared in Example 1, and sets up comparative samples, including: pure luteolin (denoted as Lut), a mixture of luteolin and copper ions (denoted as Lut+Cu, obtained by physically mixing luteolin and anhydrous CuSO4 powder), and copper ions (denoted as Cu, using anhydrous CuSO4 powder). In the experiment, the added concentration of the CuL nanocomposite was 100 μg / mL, and the Lut sample, Lut+Cu sample and Cu sample were added according to the standard of the same amount of Cu ions and / or luteolin in the composite.

[0034] 1. ABTS + Free radical scavenging experiment and DPPH free radical scavenging experiment

[0035] Using ABTS + The antioxidant properties of the samples were tested with free radical or DPPH·free radical working solution. Figure 2 As shown in (a), it can be seen from the figure that the copper ion-luteolin nanocomposite has a stronger ability to remove ABTS· + and the effects of DPPH·free radicals.

[0036] 2. OH, H2O2 (CAT-like activity), O2 - (SOD-like activity) clearance experiment

[0037] OH scavenging rate: FeSO4 (1 mM) and H2O2 (2 mM) were mixed in sodium acetate buffer, and then the CuL complex or control sample was added. After reacting for 5 min, the absorbance at 650 nm was measured using 3,3',5,5'-tetramethylbenzidine (TMB).

[0038] H2O2 scavenging rate: O2 generation capacity was assessed using a portable dissolved oxygen meter. The typical method was to add the CuL complex or control sample and H2O2 (100 μM) to pH 6.5 phosphate buffer and monitor the dissolved oxygen concentration in the system for 15 min.

[0039] ·O 2- Clearance: O2 clearance by CuL complex or control samples was assessed using a colorimetric assay kit for inhibition and generation of superoxide anions - The activity of free radicals was measured by absorbance at 450 nm.

[0040] Test results such as Figure 2 As shown in (b), it can be seen from the figure that the copper ion-luteolin nanocomposite has good ·OH, H2O2 (CAT-like), ·O2 - (SOD-like) scavenging activity.

[0041] Example 3

[0042] In this example, the copper ion-luteolin nanocomplex prepared in Example 1 was tested for its ability to remove ROS and protect cells and its immunomodulatory activity on M1 macrophages according to the following steps:

[0043] 1. RAW264.7 cells were induced with 3mM hydrogen peroxide, and the cell activity and ROS generation were detected under fluorescence confocal microscope using a cell live and dead detection kit and a DCFH-DA (2,7-Dichlorodi-hydrofluorescein diacetate) ROS detection kit. In cell live and dead staining, red fluorescence represents dead cells, and green fluorescence represents live cells; in ROS fluorescence staining, green represents ROS generated in cells, the more ROS, the stronger the green fluorescence, and blue represents the cell nucleus. The comparison samples and the added concentrations of the samples in the experiment are the same as those in Example 2.

[0044] Figure 3 (a) and Figure 3 (b) is the effect of the copper ion-luteolin nanocomposite prepared in Example 1 on scavenging ROS and protecting cells. The figure shows that a large number of cells died and ROS were produced after hydrogen peroxide induction. Compared with luteolin, copper ions and a mixture of luteolin and copper ions with equal concentrations, the copper ion-luteolin nanocomposite has better ROS scavenging ability and cell protection ability.

[0045] 2. Lipopolysaccharide (LPS) was used to stimulate macrophages to differentiate into M1 macrophages. ELISA analysis was used to evaluate the effects of copper ion-luteolin nanocomplex on the secretion of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-17A by M1 macrophages and the secretion of anti-inflammatory cytokines IL-4 and IL-10 by M2 macrophages.

[0046] Figure 4 The control group is the unactivated macrophage M0, and the LPS+CuL group is the M1 macrophage model (LPS concentration is 1μg / mL, corresponding to three concentrations of CuL: 0, 100, and 200μg / mL). It can be seen from the figure that the copper ion-luteolin nanocomplex can significantly reduce the secretion of TNF-α, IL-1β, IL-6, and IL-17A, and reduce the inflammatory response. It has a promoting effect on the secretion of anti-inflammatory cytokines IL-4 and IL-10 by M2 macrophages. Therefore, the copper ion-luteolin nanocomplex can significantly reduce the secretion of pro-inflammatory factors and promote the secretion of anti-inflammatory factors, thereby reducing the inflammatory response, and the statistical results are statistically significant, indicating that the prepared copper ion-luteolin nanocomplex can regulate the expression of innate cytokines in giant macrophages, thereby interrupting the inflammatory loop and promoting inflammatory recovery. ( * p<0.05, indicating significant difference; ** p<0.01, indicating that the difference is very significant).

[0047] Example 4

[0048] This example tests the effect of the copper ion-luteolin nanocomposite prepared in Example 1 on Nrf2 according to the following steps:

[0049] RAW264.7 cells were seeded into confocal dishes at 1 × 10 cells per well in 1 mL. 6Cells were incubated at 37°C for 12 hours. The supernatant was absorbed and the sample solution was added: 1 mL of DMEM medium was added to the Control group; 1 mL of DMEM medium containing 1 μg / mL lipopolysaccharide (LPS) was added to the LPS group; 1 mL of DMEM medium containing 1 μg / mL lipopolysaccharide (LPS) and 100 μg / mL CuL was added to the LPS+CuL group. Incubate at 37°C for 12 hours. Seal with bovine serum (BSA) for 2 hours, add permeable solution, incubate with Nrf2 antibody at 37°C for 4 hours, and rinse the cells with PBS. Then incubate with secondary antibody for 30 minutes. After adding 6-diamino-2-phenylindole 4′ (DAPI), take pictures under a fluorescence microscope. The results are as shown in the figure. Figure 5 As shown, it can be seen that the fluorescence of Nrf2 weakened after LPS induction, but the fluorescence was enhanced after the addition of copper ion-luteolin nanocomplex, indicating that copper ion-luteolin nanocomplex has a promoting effect on Nrf2.

[0050] Example 5

[0051] This example tests the preventive effect of the copper ion-luteolin nanocomposite prepared in Example 1 on inflammatory bowel disease according to the following steps:

[0052] The mice were randomly divided into the following groups: control group (Control), modeling group (DSS), blank administration group (CuL, 5mg / kg, 10mg / kg, 20mg / kg), and treatment administration group (DSS+CuL, 5mg / kg, 10mg / kg, 20mg / kg), with 4 mice in each group. The modeling method was to gavage the mice with 4% dextran sulfate sodium salt (DSS) in PBS solution to construct an inflammatory bowel disease mouse model. The prevention method was to intraperitoneally inject the mice with a PBS solution containing the treatment material.

[0053] Control group: no treatment;

[0054] Modeling group: Modeling was performed every morning for 7 consecutive days;

[0055] Blank administration group: different concentrations of copper ion-luteolin nanocomplexes were used as blank control treatment materials and administered every other day;

[0056] Preventive medication group: The copper ion-luteolin nanocomplexes of different concentrations prepared in Example 1 were used as therapeutic materials; the model was established every morning for 7 consecutive days, but medication was given every other day on the 1st, 3rd and 5th days.

[0057] The status of the mice was observed and photographed every day, and the inflammatory status of the mice in each group was scored according to the scoring criteria (Disease Activity Index of Inflammatory Bowel Disease), until the mice were euthanized, the colon was removed to measure the length and the sections were sliced ​​for H&E staining.

[0058] Figure 6 For colon pictures and colon length. Figure 6 (a) shows that the colon of the control group has no obvious phenotype, while the modeling group shows phenotypes such as blood in the stool, colon edema, and colon shortening. The preventive medication group shows significant improvement, indicating that the prepared copper ion-luteolin nanocomplex has a significant preventive effect on inflammatory bowel disease. In the pathogenesis of colitis, colon length is one of the important indicators to measure the progression of colitis. Figure 6 (b) There was no significant change in the blank administration group compared with the control group, and the colon length of the modeling group was shortened to 6.0±0.29 cm, indicating that inflammation in mice greatly shortened the colon. The simultaneous administration of copper ion-luteolin nanocomplex (5mg / kg, 10mg / kg, 20mg / kg) effectively prevented the shortening of the colon, and the three groups restored the colon length to 7.9±0.13 cm, 8.7±0.15 cm, and 9.0±0.13 cm, respectively.

[0059] Figure 7 H&E staining photos and scores. DSS-induced mice developed inflammatory symptoms, including mucosal erosion, goblet cell destruction, inflammatory cell infiltration, and crypt structure damage. In contrast, the 20 mg / kg copper ion-luteolin nanocomplex preventive administration group not only induced colon length recovery, but also had a protective effect on colon tissue. The 5 mg / kg copper ion-luteolin nanocomplex and 10 mg / kg copper ion-luteolin nanocomplex treatment groups also alleviated colon microstructure damage and cell fragmentation to a certain extent.

[0060] Figure 8 (a) is the statistical result of the weight change of mice. The weight of mice in the control group and the blank-dosing group fed only with copper ion-luteolin nanocomplex increased normally, and the weight of mice in the modeling group fed with DSS showed a significant downward trend. However, the weight loss of mice in the preventive dosing group fed with copper ion-luteolin nanocomplex was smaller, among which the weight loss of the copper ion-luteolin nanocomplex (20 mg / kg) group was the smallest, and there was an increasing trend on the 7th day. The disease activity index (DAI) is a recognized indicator of colon inflammation, which is calculated based on weight change, stool consistency index, blood and stool index. The results are shown in Figure 8As shown in (b), within 7 days, the mice in the control group and the blank treatment group that only received intraperitoneal injection of copper ion-luteolin nanocomplexes did not have loose stools or blood in the stool, and the disease activity index (DAI) score was 0, while the disease scores of the modeling group and the low-dose (5mg / kg, 10mg / kg) copper ion-luteolin nanocomplex prevention group increased significantly. The DAI index of the high-dose treatment group (20mg / kg) of copper ion-luteolin nanocomplexes increased slowly, and although slight blood in the stool appeared in the early stage, it effectively inhibited the occurrence of colitis.

[0061] Example 6

[0062] This example tests the therapeutic effect of the copper ion-luteolin nanocomposite prepared in Example 1 on inflammatory bowel disease according to the following steps:

[0063] The mice were randomly divided into the following groups: control group (Control), modeling group (DSS), and treatment group (CuL, 10 mg / kg, 20 mg / kg), with 5 mice in each group. The modeling method was to gavage the mice with 4% dextran sulfate sodium salt (DSS) in PBS solution to construct an inflammatory bowel disease mouse model. The treatment method was to intraperitoneally inject the mice with a PBS solution containing the treatment material.

[0064] Control group: no treatment;

[0065] Modeling group: Modeling was performed every morning for 7 consecutive days;

[0066] Treatment group: The copper ion-luteolin nanocomplexes of different concentrations prepared in Example 1 were used as treatment materials; after modeling in the morning for 7 consecutive days, the drugs were administered every other day on the 9th, 11th and 13th days.

[0067] The status of the mice was observed and photographed every day, and the inflammatory status of the mice in each group was scored according to the scoring criteria (disease activity index of inflammatory bowel disease), until the mice were euthanized, the colon was removed to measure the length, and the colon sections were subjected to immunohistochemical staining.

[0068] Fig. 9 (a) is a photo of the colon and the length of the colon. It can be seen from the figure that the colon of the control group has no obvious phenotype, while the model group shows phenotypes such as blood in the stool, edema, and shortening. Fig. 9(b) There was no significant change in the control group, while inflammation caused the mouse colon to shorten significantly. The simultaneous administration of copper ion-luteolin nanocomplex (10 mg / kg, 20 mg / kg) effectively treated colon inflammation, and the colon length was restored, showing a significant improvement, indicating that the prepared copper ion-luteolin nanocomplex has a significant therapeutic effect on inflammatory bowel disease.

[0069] Fig. 9 (c) is the statistical result of the weight change of mice. The weight of mice in the control group increased normally, and the weight of mice in the modeling group fed with DSS showed a significant downward trend. The weight of the treatment group treated with copper ion-luteolin nanocomplex as a therapeutic drug increased after administration, and basically recovered to the weight before modeling. The results of the disease activity index are shown in Fig. 9 As shown in (d), the disease activity index score of the mice in the control group was 0, while the disease score of the modeling group was significantly increased. The disease activity index of the copper ion-luteolin nanocomposite treatment group (10 mg / kg, 20 mg / kg) was increased in the early stage, and the increasing trend was suppressed after administration, and the disease activity index showed a downward trend, indicating that the copper ion-luteolin nanocomposite has a therapeutic effect on colon inflammation.

[0070] Fig. 9 (e) is an immunohistochemical staining photo. DSS-induced mice showed inflammatory symptoms, including a significant increase in IκKβ, P65, p-IκKβ, and p-P65. In contrast, the IκKβ, P65, p-IκKβ, and p-P65 in the 20 mg / kg copper ion-luteolin nanocomposite treatment group basically returned to normal levels, which had an inhibitory effect on colon inflammation. The 10 mg / kg copper ion-luteolin nanocomposite treatment group also alleviated colon inflammation to a certain extent.

[0071] Example 7

[0072] This embodiment tests the effect of the copper ion-luteolin nanocomposite prepared in Example 1 on the intestinal flora regulation of mice with inflammatory bowel disease:

[0073] In Example 6, after euthanizing mice, feces were collected from the cecum to analyze the abundance of bacterial flora.

[0074] Fig.10 The bar graph in (a) details the microbiome composition of each group at the phylum level, which is characterized by a significant decrease in harmful microorganisms (such as Proteobacteria) and a significant increase in beneficial bacteria (such as Bacteroidetes and Firmicutes), which support intestinal health by inhibiting harmful microorganisms and regulating mucosal immune balance. Fig.10As shown in (b), DSS-induced colitis was associated with a decrease in microbiome diversity and community richness, whereas treatment with copper ion-luteolin nanocomplexes significantly enhanced the abundance and diversity of the intestinal microbiota in mice.

[0075] In addition, the relative abundance of microbiota that varied at the family level was statistically analyzed. Potentially harmful Proteobacteria, especially Enterobacteriaceae, were reduced by CuL ( Fig.10 (c) Enterobacteriaceae can thrive in the inflamed intestine, leading to overproliferation and further exacerbating the inflammatory response associated with IBD. CuL treatment significantly increased the relative abundance of Firmicutes, especially in Helicobacteraceae and Lactobacillaceae ( Fig.10 (d) and (e)). Helicobacteraceae is associated with butyrate production, which promotes the growth of microorganisms and host epithelial cells. Bifidobacterium, a probiotic bacterium that contributes to the health of the intestinal microenvironment by competitively inhibiting harmful microorganisms and regulating intestinal immune responses, was more abundant in the CuL-treated group ( Fig.10 (f)).

[0076] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a copper ion-luteolin nanocomposite, characterized in that: The anhydrous CuSO4 and luteolin solution are uniformly dispersed in water, mixed and stirred in the presence of polyvinylpyrrolidone (PVP) to obtain a copper ion-luteolin nanocomposite.

2. The preparation method according to claim 1, characterized in that: First, PVP was dissolved in deionized water, and then anhydrous CuSO4 powder was added and stirred until clear. Then, luteolin was added and stirred at room temperature for 3 to 4 days, and then centrifuged to remove excess luteolin. The resulting supernatant was ultrafiltered to remove excess PVP and copper ions, thereby obtaining a dispersion of copper ion-luteolin nanocomplex.

3. The preparation method according to claim 1 or 2, characterized in that: The mass ratio of luteolin, anhydrous CuSO4 and PVP is 1:5-6:25-35.

4. The preparation method according to claim 2, characterized in that: The centrifugal speed is 8000-12000 rpm and the centrifugal time is 8-12 min.

5. The preparation method according to claim 2, characterized in that: The ultrafiltration used a 100 kDa ultrafiltration tube.

6. A copper ion-luteolin nanocomposite prepared by the preparation method according to any one of claims 1 to 5.

7. An application of the copper ion-luteolin nanocomposite according to claim 6, characterized in that: Used for preparing medicines for preventing and / or treating inflammatory bowel disease.

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