Inulin gel compound loaded with Prussian blue nano-enzyme and nattokinase and application of inulin gel compound in treatment of inflammatory bowel disease

Abstract

The invention discloses an inulin gel compound loaded with Prussian blue nano-enzyme and nattokinase and application of the inulin gel compound in treatment of inflammatory bowel disease, the compound is formed by blending the Prussian blue nano-enzyme, the nattokinase and inulin, the Prussian blue nano-enzyme with multiple antioxidant enzyme activities is used for removing a large amount of active oxygen in cells, and the nattokinase is used for treating inflammatory bowel disease. The level of inflammatory factors is reduced, oxidative stress is relieved, and intestinal inflammation is treated; the nattokinase not only can inhibit the formation of intestinal microthrombus and effectively dissolve thrombus through the excellent thrombolysis capability of the nattokinase, but also can synergistically and effectively treat inflammatory bowel diseases through the intestinal microbiota regulation capability of the nattokinase; in addition, the inulin, the Prussian blue nano-enzyme and the nattokinase jointly realize the regulation of intestinal microbiota of the patient with the inflammatory bowel disease, so that the anxiety and depression symptoms related to the inflammatory bowel disease are effectively relieved through microbiota-intestine-brain axis. In general, the invention provides a creative example for treating the inflammatory bowel disease and related intestinal internal and external complications thereof.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterial preparation and biomedicine, and specifically relates to a method for preparing an inulin gel complex loaded with Prussian blue nanozyme and nattokinase for treating inflammatory bowel disease and its related intestinal microthrombosis and anxiety and depression complications. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, recurrent and refractory gastrointestinal inflammatory disease that has attracted increasing public attention worldwide. The pathogenesis of IBD is still unclear, but it is generally believed to be related to factors such as high oxidative stress and dysbiosis in the intestinal microenvironment. Currently, clinically approved IBD treatment drugs such as aminosalicylate, glucocorticoids, and immunosuppressants can only temporarily relieve IBD symptoms, but cannot cure intestinal tissue damage and disordered flora. In addition, while suffering from symptoms such as diarrhea and abdominal pain caused by IBD itself, patients will also face related complications caused by long-term IBD. It is reported that the risk of venous thromboembolism (VTE) in IBD patients is 2.5 times that of the normal population. Under the stimulation of persistent inflammation at the site of intestinal tissue damage, immune cells continuously release tissue necrosis factor, promote the expression of proinflammatory tissue factor and platelet activation, form intestinal microthrombi, and even develop into more serious conditions, such as VTE, whose in situ thromboembolism can lead to intestinal ischemia and hypoxia, further aggravating intestinal inflammation and damage. Moreover, compared with the general population, IBD patients are more susceptible to anxiety, depression, and other psychiatric disorders, and the overall prevalence of depression in adult IBD patients is as high as 21%-25.2%. The imbalance of intestinal microbiota and its metabolites in IBD patients induces chronic inflammation of the intestinal epithelium, leading to the destruction of intestinal barrier integrity and increased intestinal permeability, and exacerbating neuroinflammation through the microbiota-gut-brain axis, leading to the development of psychiatric disorders including anxiety and depression. Conversely, in the state of anxiety and depression, the brain's regulation of intestinal serotonin (5-HT) and dopamine (DA) signals is disrupted, enhancing the pro-inflammatory activity of immune cells at the site of inflammation and promoting the release of inflammatory cytokines, thereby exacerbating the intestinal inflammatory response.

[0003] Reactive oxygen species (ROS) are the hallmarks and executors of oxidative stress. Excessive accumulation of ROS in the inflammatory site of the colon leads to protein oxidative damage, lipid peroxidation and DNA damage, which further leads to intestinal mucosal damage and worsening of intestinal inflammation. Prussian blue (PB), as an oral antidote approved by the United States Food and Drug Administration (USFDA) for the treatment of poisoning by radioactive elements such as thallium, has been shown to have outstanding biosafety and has become a nanozyme with great clinical translational prospects for the treatment of IBD. However, oral nanozymes have a short residence time in the gastrointestinal tract and require repeated administration to achieve the desired therapeutic effect. Repeated administration may lead to large accumulation in the body, leading to potential toxicity risks. In addition, regulating oxidative stress alone is not enough to solve the complex pathogenesis of IBD or treat IBD-related complications. Summary of the invention

[0004] The present invention develops an inulin gel complex loaded with Prussian blue nanozyme and nattokinase, which can remove reactive oxygen in intestinal inflammatory sites, regulate the homeostasis of intestinal microbiota, and treat IBD while avoiding intestinal microthrombosis and anxiety and depression symptoms caused by persistent inflammation and intestinal microbiota disorders.

[0005] In order to solve the technical problem, the present invention adopts the following technical solution:

[0006] The present invention first discloses an inulin gel complex loaded with Prussian blue nanozyme and nattokinase, which uses inulin gel as a drug-carrying system, loaded with Prussian blue nanozyme with good ability to remove reactive oxygen and nattokinase with thrombolytic ability. The use of inulin gel, on the one hand, helps the continuous release of PB nanozyme and nattokinase (NKase) in the intestine through the intestinal retention effect, thereby increasing the effective treatment time; on the other hand, it can selectively promote the proliferation of probiotics through the fermentation of intestinal microbiota, and together with NKase and PB nanozyme, achieve the regulation of intestinal microbiota of IBD patients.

[0007] The preparation method of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase of the present invention is as follows: Prussian blue nanozyme powder is dissolved in deionized water, and nattokinase is added for ultrasonic mixing, and then inulin is added and stirred until mixed evenly to obtain a reaction solution; the reaction solution is placed in a 40-45°C water bath, stirred at a constant temperature for 15-25 minutes, and then cooled at room temperature for 12-14 hours to obtain the inulin gel complex loaded with Prussian blue nanozyme and nattokinase.

[0008] Preferably, the dosage ratio of Prussian blue nanozyme, nattokinase, inulin and deionized water is 0.5-2 mg: 7.5-30 mg: 0.7 g: 1 mL. The dosage ratio of inulin to deionized water is the key to whether inulin gel can be formed.

[0009] Preferably, the rotation speed of the constant temperature stirring is 450-600 rpm.

[0010] Preferably, the preparation method of the Prussian blue nanozyme powder is as follows: 3.0 g PVP is dissolved in 30 mL 0.01 M HCL solution, 132 mg potassium ferrocyanide is added, and the mixture is stirred and ultrasonically mixed until uniformly mixed, and the resulting mixture is placed in an oven at 80° C. for 24 hours. After the reaction is completed, centrifugation, water washing, and freeze-drying are performed to obtain the Prussian blue nanozyme powder.

[0011] The inulin gel complex loaded with Prussian blue nanozyme and nattokinase of the present invention has the following characteristics: simple preparation; no significant toxicity at the cellular and animal levels; Prussian blue nanozyme can effectively remove reactive oxygen; the contained nattokinase has the ability to dissolve blood clots and regulate intestinal microflora; the intestinal retention effect of the inulin gel drug loading platform helps to continuously release Prussian blue nanozyme and nattokinase in the intestine, increase the effective treatment time, and can also selectively promote the proliferation of probiotics through fermentation of intestinal microflora and regulate the intestinal microflora.

[0012] The inulin gel complex loaded with Prussian blue nanozyme and nattokinase of the present invention can be used to prepare a medicament for regulating intestinal inflammation and intestinal microbiota to treat inflammatory bowel disease and its related intestinal microthrombosis and anxiety and depression complications. The dosage form is an oral gel. The implementation mechanism is: Prussian blue nanozyme can effectively remove excessive reactive oxygen in the colitis site through its own rich redox potential. At the same time, inulin gel can effectively protect nattokinase from the influence of gastric acid environment and prolong intestinal retention time. Nattokinase prevents intestinal microthrombosis of colitis complications by dissolving thrombus and regulating intestinal microbiota. And the inulin gel complex loaded with Prussian blue nanozyme and nattokinase can regulate the abundance and diversity of intestinal microbial communities through the coordination between the components to repair the imbalance of IBD intestinal flora, while maintaining the integrity and permeability of the intestinal barrier, thereby alleviating the anxiety and depression symptoms associated with colitis through the microbiota-intestinal-brain axis.

[0013] The beneficial effects of the present invention are embodied in:

[0014] 1. The inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared by the present invention can achieve intestinal stasis and sustained release of drugs.

[0015] 2. In the inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared by the present invention: the rich redox potential of Prussian blue nanozyme enables it to have multiple types of enzyme activities and the ability to remove multiple active oxygen species. The inherent thrombolytic ability of nattokinase enables the gel to have the ability to treat intestinal microthrombi.

[0016] 3. The synergistic effect between the components of the inulin gel complex loaded with Prussian blue nanozymes and nattokinase prepared by the present invention enables it to alleviate inflammatory bowel disease and its related intestinal and extraintestinal complications. In the preventive and delayed colitis models induced by DSS (dextran sulfate sodium), the gel complex of the present invention significantly alleviated the occurrence of enteritis; in the intestinal microthrombosis complication model, it significantly reduced the formation of thrombus; and significantly alleviated the anxiety and depression caused by long-term chronic colitis.

[0017] 4. The inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared by the present invention has a simple preparation method, mild reaction conditions, low production cost, good injectability and biosafety, and has potential for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the synthesis of an inulin gel complex containing Prussian blue nanozyme and nattokinase according to the present invention.

[0019] Figure 2 This is a transmission electron microscopy image of the Prussian blue nanozyme prepared in Example 1.

[0020] Figure 3 This is the XRD (X-ray diffraction) spectrum of the Prussian blue nanozyme prepared in Example 1.

[0021] Figure 4 This is the rheological diagram of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared in Example 1.

[0022] Figure 5 This is a scanning electron micrograph of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared in Example 1.

[0023] Figure 6 This is an injectable image of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared in Example 1.

[0024] Figure 7 This is a graph showing the in vitro scavenging of ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) free radicals by the inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared in Example 1.

[0025] Figure 8 This is a comparison chart of the retention of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase prepared in Example 1 and the simple Prussian blue nanozyme in the intestine, wherein: Figure 8(a) The distribution of materials in the intestinal tract of mice at different time points after oral administration of pure Cy5.5-labeled Prussian blue nanozyme and inulin gel complex loaded with Cy5.5-labeled Prussian blue nanozyme and nattokinase, as photographed by small animal imaging software; Figure 8 (b) is obtained through small animal imaging software Figure 8 (a) is a comparison chart of the results of quantitative analysis of fluorescence intensity.

[0026] Fig. 9 The in vitro thrombolysis diagram of each group in the in vitro thrombolytic performance test of Example 1, wherein: Fig. 9 (a) is a photo of a thrombus. Fig. 9 (b) Quantification of thrombus dissolution rate.

[0027] Fig.10 The test results of each group in the colitis prevention and treatment performance test of Example 1, wherein: Fig.10 (a) is the weight change of mice. Fig.10 (b) is a colon photo. Fig.10 (c) is a statistical chart of colon length.

[0028] Fig.11 These are the test results of each group in the delayed treatment performance test of colitis in Example 1, wherein: Fig.11 (a) is the disease activity index of mice, Fig.11 (b) is a colon photo. Fig.11 (c) is a statistical chart of colon length.

[0029] Fig.12 These are images of bloody stools of mice in each group in the intestinal microthrombosis regulation performance test of Example 1.

[0030] Fig.13 This is the elevated plus maze test in the performance test of relieving anxiety and depression symptoms caused by colitis in Example 1, wherein: Fig.13 (a) Elevated trajectory diagram, Fig.13 (b) open arm time, Fig.13 (c) Number of open arm crossings. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail in combination with the embodiments below. This embodiment is implemented on the premise of the technical scheme of the present invention, and a detailed implementation mode and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0032] Example 1

[0033] In this example, the inulin gel complex loaded with Prussian blue nanozyme and nattokinase was prepared as follows:

[0034] (1) Preparation of Prussian blue nanozyme: 3.0 g PVP was dissolved in 30 mL 0.01 M HCL solution, 132 mg potassium ferrocyanide was added, and the mixture was stirred and ultrasonicated until uniformly mixed. The resulting mixture was placed in an oven at 80°C for 24 h. After the reaction, the mixture was centrifuged at 14,000 rpm for 10 min, washed with water 5 times, and freeze-dried to obtain Prussian blue nanozyme powder.

[0035] (2) Preparation of inulin gel complex loaded with Prussian blue nanozyme and nattokinase Figure 1 As shown: 2 mg of the Prussian blue nanozyme prepared in step (1) was weighed and dissolved in 1 mL of deionized water, and 10 mg of nattokinase was weighed and added thereto for ultrasonic mixing, followed by adding 0.7 g of inulin and stirring until mixed uniformly to obtain a reaction solution. The reaction solution was placed in a 45°C water bath, stirred at a constant temperature of 450 rpm for 20 min, and then allowed to stand and cool at room temperature for 12 h to obtain an inulin gel complex loaded with Prussian blue nanozyme and nattokinase (denoted as PB / NKase@Inulin gel).

[0036] For comparison, this embodiment also prepared pure inulin gel (denoted as Inulin gel), inulin gel loaded only with Prussian blue nanozyme (denoted as PB@Inulin gel), and inulin gel loaded only with nattokinase (denoted as NKase@Inulin gel). The preparation method was the same as that of PB / NKase@Inulin gel, and the only difference was that the amount of Prussian blue nanozyme and nattokinase added was adjusted (when preparing pure inulin gel, the amount of both added was 0; when preparing PB@Inulin gel, the PB nanozyme was kept at 2 mg and the amount of nattokinase added was 0; when preparing NKase@Inulin gel, the amount of PB nanozyme added was 0 and the amount of nattokinase added was kept at 10 mg).

[0037] The inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example was characterized in terms of morphology and performance as follows:

[0038] 1. Morphological Characterization

[0039] Figure 2 This is a transmission electron micrograph of the Prussian blue nanozyme obtained in this example. From the figure, it can be seen that the diameter of the nanozyme is 200-300 nm.

[0040] Figure 3This is the XRD (X-ray diffraction) spectrum of the Prussian blue nanozyme obtained in this example. It can be seen from the figure that only the characteristic peak of Prussian blue is displayed, and there are no other heterogeneous peaks, indicating that the Prussian blue nanozyme is successfully synthesized.

[0041] Figure 4 This is the rheological diagram of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example. From the figure, it can be seen that the storage modulus (G') of the inulin gel complex is greater than the loss modulus (G"), which is consistent with the rheological characteristics of the colloid, and the inulin gel complex is successfully synthesized.

[0042] Figure 5 This is a scanning electron micrograph of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example. It can be observed from the figure that the inulin gel complex has an ordered porous structure.

[0043] By regulating the amount of Prussian blue nanozyme and / or nattokinase added, the mass ratio of Prussian blue nanozyme and nattokinase in the prepared complex can be regulated so that the two can work together to achieve the optimal effect. It has been verified that in a system of 0.7g inulin and 1mL deionized water, gel can be formed when the amount of Prussian blue nanozyme added is 0.5-2mg and the amount of nattokinase added is 7.5-30mg.

[0044] The mass of Prussian blue nanozyme was adjusted to 1 mg, and the mass of nattokinase was adjusted to 15 mg, to prepare an inulin gel complex loaded with Prussian blue nanozyme and nattokinase, which was then loaded into syringes of different sizes and then injected, such as Figure 6 As shown in the figure, it can be seen that the inulin gel complex can be injected through syringe needles of different sizes, with the smallest being only 0.4 mm, and has excellent injectability.

[0045] 2. In vitro ABTS removal performance test

[0046] Figure 7The in vitro scavenging diagram of ABTS (2,2'-azo-bis-3-ethylbenzothiazoline-6-sulfonic acid) free radical of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example. ABTS is a relatively stable artificial synthetic free radical, which shows a blue-green color and has a characteristic ultraviolet absorption peak at a wavelength of 734nm. The effect of scavenging ABTS is detected by the ultraviolet absorption peak at 734nm after the detection material is co-incubated with ABTS. The characterization method is: accurately weigh 38.4mgABTS and 19.2mg potassium persulfate, dissolve them in 10mL deionized water, shake them well and place them in the dark for 12-16 hours to allow ABTS to fully oxidize and generate ABTS free radical cations. The solution after the reaction is the ABTS free radical stock solution, and its color should be blue-green. The ABTS stock solution is diluted with PBS (pH=7.4) so ​​that the absorbance value at a wavelength of 734nm is in the range of 0.7-0.8 to obtain an ABTS working solution. Different masses of inulin gel complexes containing Prussian blue nanozymes and nattokinase (prepared by the system of "2 mg Prussian blue nanozymes + 10 mg nattokinase + 0.7 g inulin + 1 mL deionized water") were added to 2 mL ABTS working solution, so that the final concentration of nattokinase in the system was 5, 10, 20, 40 or 80 μg / mL, respectively, and incubated in the dark for 5 min. After the incubation was completed, centrifuged at 14000 rpm for 10 min, the supernatant was taken, and the ultraviolet absorption at 734 nm was measured. As can be seen from the figure, the ABTS scavenging capacity when the inulin gel complex was added to the nattokinase concentration of 80 μg / mL was about 80%.

[0047] 3. Intestinal retention performance test

[0048] Figure 8The retention comparison diagram of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example and the simple Prussian blue nanozyme in the intestine. The specific experimental steps are: first weigh 2 mg of Prussian blue nanozyme and add 1 mL of deionized water, add 20 μL of 0.2 mg / mL Cy5.5 after ultrasonic dispersion, incubate at 37 ° C for 12 hours, and then dialyze with a 14000Da dialysis bag for 24 hours to obtain Cy5.5-labeled Prussian blue nanozyme. Then, according to the method of Example 1, the inulin gel complex loaded with Prussian blue nanozyme and nattokinase was prepared with Cy5.5-labeled Prussian blue nanozyme, and the mice were orally gavaged with simple Cy5.5-labeled Prussian blue nanozyme and inulin gel complex loaded with Cy5.5-labeled Prussian blue nanozyme and nattokinase. The mice were dissected at 4, 8, 12, and 24 hours, and the stomach to the colon was taken, and the fluorescence distribution was photographed using small animal imaging software, and the fluorescence intensity was quantitatively analyzed. As can be seen from the figure, the simple Cy5.5-labeled Prussian blue nanozyme reached its peak at 8 hours, while the inulin gel complex loaded with Cy5.5-labeled Prussian blue nanozyme and nattokinase reached its peak at 12 hours, and at 24 hours, the inulin gel complex loaded with Cy5.5-labeled Prussian blue nanozyme and nattokinase had a stronger fluorescence intensity and retained in the colon for a longer time.

[0049] IV. In vitro thrombolytic performance test

[0050] Fig. 9 This is a comparison chart of the in vitro thrombolysis of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example. The characterization method is as follows: prepare the following different groups of materials: (1) PBS group; (2) inulin gel group (Inulin gel, 0.7 g / mL inulin); (3) Prussian blue nanozyme group (PB NZs, 2 mg / mL); (4) inulin gel group containing Prussian blue nanozyme (PB@Inulin gel, 2 mg / mL Prussian blue nanozyme, 0.7 g / mL inulin); (5) nattokinase group (NKase, 10 mg / mL); (6) inulin gel group containing nattokinase (NKase@Inulin gel, 10 mg / mL nattokinase, 0.7 g / mL inulin); (7) inulin gel group containing Prussian blue nanozyme and nattokinase (PB / NKase@Inulin gel, 2 mg / mL Prussian blue nanozyme, 10 mg / mL nattokinase, 0.7 g / mL inulin). Rabbit blood was collected in a culture dish by the heart blood sampling method, and the supernatant was discarded after being placed at room temperature for 4 hours to obtain rabbit blood clots. The rabbit blood clots were divided into equal sizes and incubated with different groups of materials for 0, 1 or 3 hours, and the blood clots were observed and weighed. Fig. 9 (a) It can be seen that the volume of thrombus in groups (5), (6), and (7) containing nattokinase was significantly reduced. Fig. 9 (b) It can be seen that the in vitro nattokinase group has the best thrombolytic performance.

[0051] 5. Colitis prevention and treatment performance

[0052] The inulin gel complex was prepared according to the dosage. In this test, when the dosage of Prussian blue nanozyme was 5 mg / kg and the dosage of nattokinase was 75 mg / kg, it was prepared by the "0.5 mg Prussian blue nanozyme + 7.5 mg nattokinase + 0.7 g inulin + 1 mL deionized water" system. At this time, the dosage of inulin gel was 7 g / kg.

[0053] Fig.10 The statistical graph is a result of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example after preventing and treating colitis mice. The characterization method is: 5-6 week old female C57BL / 6J normal mice were randomly divided into eight groups (n=8 per group) after one week of adaptation. The dosage types of each group are as follows: (1) Control group: PBS + normal water; (2) DSS group: PBS + 3% DSS (3) Inulin gel: inulin gel (7 g / kg) + 3% DSS; (4) PB NZs group: Prussian blue nanozyme (5 mg / kg) + 3% DSS; (5) PB@Inulin gel: inulin gel containing Prussian blue nanozyme (5 mg / kg) + 3% DSS; (6) NKase group: nattokinase (75 mg / kg) + 3% DSS; (7) NKase@Inulin gel: inulin gel containing nattokinase (75 mg / kg) + 3% DSS; (8) PB / NKase@Inulin gel: inulin gel containing Prussian blue nanozyme (5 mg / kg) and nattokinase (75 mg / kg) + 3% DSS.

[0054] The mice in the experimental group were fed with 3% DSS for seven consecutive days, and the mice in the drug-treated group were gavaged on days 1, 3, 5, and 7. The weight changes of the mice were recorded every day. On the ninth day, the mice were euthanized, and their colons were dissected and the colon lengths of each group of mice were recorded. Fig.10 (a) shows the weight changes of each group during the experiment. It can be seen that DSS caused the mice to lose weight continuously. After treatment, it can be found that the weight loss trend was alleviated. After treatment with the inulin gel complex loaded with Prussian blue nanozyme and nattokinase, the weight can be restored to normal level close to the control group. Fig.10 (b) Colonography and Fig.10 (c) The colon length statistics show that DSS treatment caused the colon of mice to shorten significantly. The colon length of mice in the treatment groups recovered to a certain extent, among which the inulin gel group loaded with Prussian blue nanozyme and nattokinase was closest to the colon length of mice in the normal group.

[0055] 6. Delayed treatment performance of colitis

[0056] The inulin gel complex used in this test was prepared according to the same system as the "colitis preventive therapeutic performance".

[0057] Fig.11 The statistical graph of the results of delayed treatment of colitis mice by the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example. The characterization method is: 5-6 week old female C57BL / 6J normal mice were randomly divided into eight groups (n=8 per group) after one week of adaptation. The dosage types of each group are as follows: (1) Control group: PBS + normal water; (2) DSS group: PBS + 3% DSS (3) Inulin gel: inulin gel (7 g / kg) + 3% DSS; (4) PB NZs group: Prussian blue nanozyme (5 mg / kg) + 3% DSS; (5) PB@Inulin gel: inulin gel containing Prussian blue nanozyme (5 mg / kg) + 3% DSS; (6) NKase group: nattokinase (75 mg / kg) + 3% DSS; (7) NKase@Inulin gel: inulin gel containing nattokinase (75 mg / kg) + 3% DSS; (8) 5-ASA group: 5-aminosalicylic acid (75 mg / kg) + 3% DSS; (9) PB / NKase@Inulingel: inulin gel containing Prussian blue nanozyme (5 mg / kg) and nattokinase (75 mg / kg) + 3% DSS.

[0058] The mice in the experimental group were fed 3% DSS from day 0 to day 7, and then switched to drinking water after day 7. Each mouse was gavaged with the corresponding material on days 7, 9, 13, and 15, and the fecal viscosity and fecal bleeding of the mice were observed every day to record the disease activity index of the mice. On day 15, all mice were euthanized, dissected, and their colons were removed and their lengths were measured. Fig.11 (a) The results of the disease activity index of mice showed that the disease activity index of mice treated with the inulin gel complex loaded with Prussian blue nanozyme and nattokinase began to decrease on the 7th day. On the 15th day, it returned to a level close to that of the normal group. Fig.11 (b) Colonography and Fig.11 (c) Colon length statistics show that the colon lengths of groups (7), (8), and (9) are significantly longer than those of the other DSS-treated groups and are similar to those of the Control group. This confirms that the inulin gel complex loaded with Prussian blue nanozymes and nattokinase has excellent delayed treatment performance for colitis.

[0059] 7. Test on the performance of regulating intestinal microthrombosis

[0060] The inulin gel complex was prepared according to the dosage. In this test, when the dosage of Prussian blue nanozyme was 5 mg / kg and the dosage of nattokinase was 150 mg / kg, it was prepared by the "0.5 mg Prussian blue nanozyme + 15 mg nattokinase + 0.7 g inulin + 1 mL deionized water" system. At this time, the dosage of inulin gel was 7 g / kg.

[0061] Fig.12 This is an image of blood in the stool of mice after the inulin gel complex loaded with Prussian blue nanozyme and nattokinase regulated intestinal microthrombosis obtained in this example. The characterization method is as follows: 5-6 week old female C57BL / 6J normal mice were randomly divided into eight groups (n=8 per group) after one week of adaptation. The drug administration types of each group are as follows: (1) Control group: PBS + normal water; (2) DSS group: PBS + 4% DSS (3) Inulin gel: inulin gel (7 g / kg) + 4% DSS; (4) PB NZs group: Prussian blue nanozyme (5 mg / kg) + 4% DSS; (5) PB@Inulin gel: inulin gel containing Prussian blue nanozyme (5 mg / kg) + 4% DSS; (6) NKase group: nattokinase (150 mg / kg) + 4% DSS; (7) NKase@Inulin gel: inulin gel containing nattokinase (150 mg / kg) + 3% DSS; (8) PB / NKase@Inulin gel: inulin gel containing Prussian blue nanozyme (5 mg / kg) and nattokinase (150 mg / kg) + 3% DSS;

[0062] The experimental group received 4% DSS instead of drinking water to induce intestinal microthrombosis from day 0 to day 7, and gavage the corresponding materials every day. After seven days, 4% DSS was replaced with drinking water, and the blood in the anus of the mice was photographed on day 9. Fig.12 As shown, the DSS group showed obvious blood in the stool, while the inulin gel groups containing nattokinase (7) and (8) showed almost no blood in the stool, which was similar to the Control group.

[0063] 8. Performance test on relieving anxiety and depression symptoms caused by colitis

[0064] The inulin gel complex used in this test was prepared according to the same system as the "colitis preventive therapeutic performance".

[0065] Fig.13 This is an elevated plus maze experiment on the effect of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase obtained in this example on relieving anxiety and depression symptoms caused by colitis. Fig.13 (a) Elevated trajectory diagram, Fig.13 (b) Arm opening time and Fig.13(c) Open arm crossing times. The characterization method is as follows: 5-6 week old male C57BL / 6J mice were randomly divided into 8 groups (n=10) after one week of adaptation. The drug administration type of each group is as follows: (1) Control group: PBS; (2) DSS group: PBS (3) Inulin gel: inulin gel (7g / kg); (4) PB NZs group: Prussian blue nanozyme (5mg / kg); (5) PB@Inulingel: inulin gel containing Prussian blue nanozyme (5mg / kg); (6) NKase group: nattokinase (75mg / kg); (7) NKase@Inulin gel: inulin gel containing nattokinase (75mg / kg); (8) PB / NKase@Inulin gel: inulin gel containing Prussian blue nanozyme (5mg / kg) and nattokinase (75mg / kg). The construction of DSS-induced chronic colitis involves the administration of three cycles of DSS. Each cycle consisted of one week of drinking 2% DSS followed by two weeks of drinking water. In the second and third cycles of water treatment, mice were gavaged with different groups of materials every other day for a total of 14 times. The control group drank water throughout the treatment period and received PBS gavage during the drug administration period.

[0066] After DSS-induced chronic colitis was successfully established, the elevated plus maze was used to quantify the anxiety behavior of mice: a maze with two open arms and two closed arms arranged vertically and crossed was used to assess the anxiety-like behavior of mice. The maze itself was 50 cm above the ground. The mice were placed in the center, during which the movement of the mice was recorded using video tracking software. The number of times the mice passed through the open arms and the length of time they stayed in the open arms were used to measure the anxiety level within a 5-minute time frame.

[0067] Fig.13 (a) shows the movement trajectory of each group of mice. From the movement trajectories of different groups, it can be seen that DSS caused the movement trajectory and range of mice in the open arms to be significantly reduced, and the exercise ability was also significantly reduced, which indicates that the mice developed anxiety behavior due to the influence of colitis. Fig.13 (b, c) show that after treatment with the inulin gel complex loaded with Prussian blue nanozyme and nattokinase, the number of open arm crossings and activity time of mice were significantly higher than those of the DSS group, the motor ability of mice was significantly restored, and the behavioral cognitive ability of the treated mice was significantly improved compared with the DSS group. The above results show that the inulin gel complex obtained in this example can relieve the anxiety and depression symptoms caused by colitis.

[0068] The above are merely exemplary 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. An inulin gel complex loaded with Prussian blue nanozyme and nattokinase, characterized in that: The gel complex is formed by mixing Prussian blue nanozyme, nattokinase and inulin.

2. The inulin gel complex loaded with Prussian blue nanozyme and nattokinase according to claim 1, characterized in that: The inulin gel complex loaded with Prussian blue nanozyme and nattokinase is injectable.

3. A method for preparing the inulin gel complex loaded with Prussian blue nanozyme and nattokinase according to claim 1 or 2, characterized in that: The Prussian blue nanozyme powder was dissolved in deionized water, and nattokinase was added and ultrasonically mixed, and then inulin was added and stirred until mixed to obtain a reaction solution; the reaction solution was placed in a 40-45°C water bath, stirred at a constant temperature for 15-25 minutes, and then cooled at room temperature for 12-14 hours to obtain an inulin gel complex loaded with Prussian blue nanozyme and nattokinase.

4. The preparation method according to claim 3, characterized in that: The dosage ratio of Prussian blue nanozyme, nattokinase, inulin and deionized water is 0.5-2 mg: 7.5-30 mg: 0.7 g: 1 mL.

5. The preparation method according to claim 3, characterized in that: The preparation method of the Prussian blue nanozyme powder is as follows: 3.0 g of PVP is dissolved in 30 mL of 0.01 M HCL solution, 132 mg of potassium ferrocyanide is added, and the mixture is stirred and ultrasonically mixed until uniformly mixed, and the resulting mixture is placed in an oven at 80° C. for reaction for 24 hours; after the reaction is completed, centrifugation, water washing, and freeze-drying are performed to obtain the Prussian blue nanozyme powder.

6. The preparation method according to claim 3, characterized in that: The rotation speed of the constant temperature stirring is 450-600 rpm.

7. An application of the inulin gel complex loaded with Prussian blue nanozyme and nattokinase according to claim 1 or 2, characterized in that: Used to prepare a medicament for regulating intestinal inflammation and intestinal microbiota to treat inflammatory bowel disease and its related intestinal microthrombosis and anxiety and depression complications.

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