Chloroplast-loaded tea polyphenol self-polymerization nanoparticles as well as preparation method and application thereof

Abstract

The invention provides chloroplast-loaded tea polyphenol self-polymerization nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of nano materials. The preparation method comprises the following steps: (1) cleaning spinach, crushing, and centrifuging to obtain chloroplast; and (2) respectively dropwise adding an epigallocatechin gallate solution and a manganese sulfate monohydrate solution into the chloroplast solution, and shaking in a shaking table, so as to obtain the tea polyphenol self-polymerized nanoparticles, which are named as EGCG-Mn coated Chloroplast. The prepared tea polyphenol self-polymerized nanoparticles can effectively improve pathological injury of colon tissue, have a good relieving effect on pyoptosis of kidneys while acting in intestinal tracts to regulate and control inflammation, and provide a new potential strategy for intervention of enteritis-related kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a chloroplast-supported tea polyphenol self-polymerized nanoparticle, its preparation method, and its application. Background Technology

[0002] Tea is one of China's traditional beverages. In Chinese tea culture, drinking tea is believed to invigorate the spirit and prolong life, a fact supported by modern science. The anti-aging components in tea are primarily tea polyphenols (TP), a collective term for a series of polyphenolic compounds in tea, mainly catechins, including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). Related studies have shown that tea polyphenols and products enriched with them have significant effects in preventing tumors (Annual Review of Pharmacology and Toxicology, 2002; 42:25-54), cardiovascular diseases (Circulation, 2004; 109:2448-2453), and cancer (Molecules, 2007, 12, 946). Meanwhile, catechins possess excellent antioxidant and metal ion chelating abilities, thus tea polyphenols are considered promising bio-based monomers. At the same time, nanomaterials, due to their microscopic properties, have broad application prospects in the energy, health, and environmental fields. Considering these two points, the topic of constructing functionalized polymer nanomaterials using tea polyphenols as monomers warrants further investigation.

[0003] For example, Chinese patent CN116585343A discloses a nucleic acid-loaded tea polyphenol self-polymerized nanoparticle, comprising tea polyphenol nanoparticles and nucleic acid drugs. The tea polyphenol nanoparticles are formed by the self-polymerization of epigallocatechin gallate in a weakly alkaline environment via the catalytic oxidation of divalent manganese ions. During the oxidative self-polymerization process, the tea polyphenol nanoparticles encapsulate nucleic acid drugs through polymerization, and the nucleic acid loading rate of the tea polyphenol nanoparticles is higher than 80%. The preparation method is simple: epigallocatechin gallate and nucleic acid are dissolved together in a weakly alkaline buffer solution, and divalent manganese ions are added to catalyze the oxidative self-polymerization. The nanoparticles have a negatively charged surface, making them less prone to adsorbing protein crowns during in vivo transport, thus avoiding rapid clearance during in vivo circulation. They possess good nucleic acid drug protection capabilities, and after transfection into cells, they can successfully release nucleic acids and exert biological functions, effectively reducing intracellular oxidative stress and inflammation levels, showing promising application prospects.

[0004] For example, Chinese patent CN107312139A discloses an antioxidant polyphenol nanomaterial, which is prepared from tea polyphenols and theophylline in a mass ratio of 2 parts by weight of tea polyphenols and 0.5-2 parts by weight of theophylline. This invention provides a pure organic antioxidant polyphenol nanomaterial and its preparation method without the use of exogenous additives. The particle size of the product can be adjusted by modifying the formula, and the particle size of the obtained nanomaterial is in the range of 100-300 nm. Furthermore, the antioxidant polyphenol nanomaterial obtained by this invention exhibits excellent free radical scavenging properties and is expected to be widely used as a green antioxidant and a safe and non-toxic food additive.

[0005] However, the existing nanomaterials or nanoparticles containing tea polyphenols have low loading rates and poor particle size uniformity. Furthermore, there are no existing technologies for loading chlorophyll onto tea polyphenol self-polymerized nanoparticles and their preparation methods. Therefore, there is an urgent need to develop a chloroplast-loaded tea polyphenol self-polymerized nanoparticle, its preparation method, and its application. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to provide a chloroplast-loaded tea polyphenol self-polymerized nanoparticle, its preparation method and application.

[0007] The morphology of the chloroplast-loaded tea polyphenol self-polymerized nanoparticles prepared by this invention is an irregularly shaped sphere with chloroplasts encapsulated inside the nanoparticles. This helps the chloroplasts overcome gastric acid and better reach intestinal lesions to exert their effects. Furthermore, the nanoparticles can exist stably in intestinal fluid.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] On one hand, this invention provides a method for preparing chloroplast-loaded tea polyphenol self-polymerized nanoparticles, the steps of which are: (1) After washing the spinach, crush it and centrifuge it to obtain chloroplasts; (2) First, add epigallocatechin gallate solution (EGCG) to the chloroplast solution, then add manganese sulfate monohydrate solution (MnSO4·H2O), shake on a shaker, and the tea polyphenol self-polymerized nanoparticles are obtained, named EGCG-Mn@Chloroplast.

[0010] The concentration of chloroplasts in the chloroplast solution mentioned in step (2) is 2-3 mg / mL; More preferably, the concentration of chloroplasts in the chlorophyll solution described in step (2) is 2.5 mg / mL.

[0011] The concentration of the epigallocatechin gallate solution mentioned in step (2) is 80-120 ug / mL; Preferably, the concentration of the epigallocatechin gallate solution in step (2) is 90-110 ug / mL.

[0012] More preferably, the concentration of the epigallocatechin gallate solution in step (2) is 100 ug / mL.

[0013] The concentration of the manganese sulfate monohydrate solution mentioned in step (2) is 80-120 ug / mL; Preferably, the concentration of the manganese sulfate monohydrate solution in step (2) is 90-110 ug / mL.

[0014] More preferably, the concentration of the manganese sulfate monohydrate solution in step (2) is 100 μg / mL.

[0015] The volume ratio of the chloroplast solution, epigallocatechin gallate solution, and manganese sulfate monohydrate solution is 2:1:2.

[0016] The shaking temperature of the shaker in step (2) is 4℃, the rotation speed is 150-250rpm, and the time is 2-4h.

[0017] Preferably, the shaking temperature of the shaker is 4°C, the rotation speed is 200 rpm, and the shaking time is 3 hours.

[0018] On the other hand, the present invention provides a tea polyphenol self-polymerized nanoparticle carrying chloroplasts prepared by the above preparation method.

[0019] In another aspect, the present invention provides the application of the above-mentioned chloroplast-loaded tea polyphenol self-polymerized nanoparticles in the preparation of drugs for treating inflammatory bowel disease.

[0020] Preferably, the inflammatory enteritis is colitis; more preferably, the colitis is colitis induced by sodium dextran sulfate.

[0021] In another aspect, the present invention provides the application of the above-mentioned chloroplast-loaded tea polyphenol self-polymerized nanoparticles in the preparation of a drug for treating enteritis and nephritis.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The tea polyphenol self-polymerized nanoparticles provided by the present invention are formed by encapsulating chlorophyll during the catalytic oxidation self-polymerization process of EGCG and MnSO4·H2O. TEM electron microscopy shows that the morphology of EGCG-Mn@Chloroplast is an irregular sphere. SEM electron microscopy shows that EGCG-Mn aggregates on the surface of chloroplasts, which can help chloroplasts overcome gastric acid and better reach intestinal lesions to exert their effects. The structure is simple and stable, with no obvious toxic side effects and high biosafety. (2) The tea polyphenol self-polymerized nanoparticles provided by this invention have the effect of inhibiting ABTS, SOD, hydroxyl radicals (•OH) and superoxide anions (O2). - All of them showed high scavenging effects, indicating that EGCG-Mn@Chloroplast can effectively neutralize a variety of reactive oxygen species and has excellent free radical scavenging ability and in vitro antioxidant capacity.

[0023] (3) The tea polyphenol self-polymerized nanoparticles provided by the present invention have excellent therapeutic effects on DSS-induced colitis in mice and have significant repair effects in the DSS-induced colon injury model in mice, which can effectively improve the pathological damage of colon tissue.

[0024] (4) The tea polyphenol self-polymerized nanoparticles provided by the present invention not only regulate inflammation in the intestine, but also have a good alleviating effect on kidney pyogenicity.

[0025] The results of serum inflammatory factor detection show that EGCG-Mn@Chloroplast exerts a significant therapeutic effect on DSS-induced kidney inflammation by inhibiting systemic inflammatory response and improving renal metabolic function, providing a new potential strategy for intervention of enteritis-related kidney injury. Attached Figure Description

[0026] Figure 1 Schematic diagram of the synthesis of chloroplast polyphenol aggregates; Figure 2 Electron micrographs and potential maps of EGCG-Mn@Chloroplast prepared in Example 1, wherein A is a scanning electron microscope image of EGCG-Mn@Chloroplast, scale bar = 2 μm; B is a transmission electron microscope image of EGCG-Mn@Chloroplast, scale bar = 1 μm; C is a zeta potential map of EGCG-Mn@Chloroplast. Figure 3 Figure 1 shows the results of free radical scavenging and in vitro antioxidant capacity tests. AD represents the in vitro antioxidant capacity of EGCG-Mn@Chloroplast, and EG represents the ESR spectrum of the free radical adduct captured by EGCG-Mn@Chloroplast. Figure 4 The treatment effect diagram shows that A is the drug administration flowchart for mice; B is the weight change curve of mice; and C is the DAI score curve of mice. Figure 5 Colonic hematoxylin and eosin (H&E) staining images, scale bar = 200 μm; Figure 6Relative expression levels; where A represents the relative expression level of mRNA Occludin; B represents the relative expression level of mRNA E-Cadherin; C represents the relative expression level of mRNA IL-1β; D represents the relative expression level of mRNA NLRP3; E represents the relative expression level of mRNA Caspase 1; and F represents the relative expression level of mRNA GSDMD. p < 0.05 p < 0.01, p < 0.001, p < 0.0001; Figure 7 Relative expression level diagram; where A represents the relative expression level of mRNA GSDMD; B represents the relative expression level of mRNA IL-18; p < 0.05 p < 0.01, p < 0.001, p < 0.0001; Figure 8 Graphs showing the expression levels of serum inflammatory factors and renal function markers; p < 0.05 p < 0.01, p < 0.001 p < 0.0001; Figure 9 Results and analysis of flow cytometry on mouse spleen; p < 0.05 p < 0.01, p < 0.001, p < 0.0001. Detailed Implementation

[0027] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0028] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0029] The method for determining chloroplast concentration in this invention is as follows: The absorbance at 652 nm is measured using a UV spectrophotometer; Chloroplast concentration = (A... 652 / 34.5)×30 (mg / ml).

[0030] Example 1: A method for preparing tea polyphenol self-polymerized nanoparticles carrying chloroplasts The steps are as follows: (1) After washing the spinach, crush and centrifuge it to obtain chloroplasts, and prepare it into a solution with a concentration of 2.5 mg / mL; (2) Add 1 mL of 100 ug / mL epigallocatechin gallate solution (EGCG) and 2 mL of 100 ug / mL manganese sulfate monohydrate solution (MnSO4·H2O) to 2 mL of chloroplast solution, and shake in a shaker at 4℃ and 200 rpm for 3 h to obtain the tea polyphenol self-polymerized nanoparticles, named EGCG-Mn@Chloroplas.

[0031] Comparative Example 1: A method for preparing chloroplast-supported tea polyphenol self-polymerized nanoparticles The steps are as follows: (1) After washing the spinach, crush and centrifuge it to obtain chloroplasts, and prepare a solution with a concentration of 1.5 mg / mL. (2) Add 1 mL of 80 ug / mL epigallocatechin gallate solution (EGCG) and 2 mL of 80 ug / mL manganese sulfate monohydrate solution (MnSO4·H2O) to 2 mL of chloroplast solution, and shake in a shaker at 4℃ and 150 rpm for 2 h to obtain the tea polyphenol self-polymerized nanoparticles, named EGCG-Mn@Chloroplast-1.

[0032] Comparative Example 2: A method for preparing chloroplast-supported tea polyphenol self-polymerized nanoparticles The steps are as follows: (1) After washing the spinach, crush and centrifuge it to obtain chloroplasts, and prepare it into a solution with a concentration of 4 mg / mL; (2) Add 1 mL of 120 ug / mL epigallocatechin gallate solution (EGCG) and 2 mL of 120 ug / mL manganese sulfate monohydrate (MnSO4·H2O) to 2 mL of chloroplast solution, and shake in a shaker at 4℃ and 250 rpm for 4 h to obtain the tea polyphenol self-polymerized nanoparticles, named EGCG-Mn@Chloroplast-2.

[0033] Comparative Example 1: After the synthesis of EGCG-Mn@Chloroplast-1, the material was subjected to acetone pyrolysis. The absorbance was measured at a wavelength of 652 nm and calculated. It was found that due to the low initial concentration of chloroplasts and the short reaction time, the final material concentration was insufficient, and the yield of chloroplast-tea polyphenol self-polymerized nanoparticles was low. When this material was used in an in vitro antioxidant kit for detection, its free radical scavenging rate was also correspondingly low.

[0034] Comparative Example 2: During the synthesis of EGCG-Mn@Chloroplast-2, the excessively long reaction time caused the material solution to freeze. After pyrolysis with acetone, the absorbance was measured and calculated at a wavelength of 652 nm. The results showed that the initial concentration of chloroplasts was high, but when used in an in vitro antioxidant kit, its free radical scavenging ability was almost completely lost. It is speculated that this is because the freezing of the solution severely damaged the structure of the chloroplasts, causing them to completely lose their original antioxidant and free radical scavenging abilities.

[0035] Example 1: During the synthesis of EGCG-Mn@Chloroplast, the reaction conditions were well-suited, and no problems such as solution freezing or excessive precipitation occurred. After acetone pyrolysis, the absorbance of the material was measured and calculated at 652 nm. The results showed that the chloroplast concentration was moderate, the material concentration met the target, and the yield was excellent. When used in an in vitro antioxidant kit, the free radical scavenging rate reached the optimal level. This is presumably because the optimized reaction parameters ensured the complete preservation of the chloroplast structure while promoting the synthesis of EGCG and Mn. 2+ The binding efficiency with chloroplasts ultimately maximizes the material's antioxidant and free radical scavenging capabilities.

[0036] The self-polymerized tea polyphenol nanoparticles carrying chloroplasts prepared in Example 1 were subjected to [further action / action], based on [the specific details]. Figure 2 TEM images of EGCG-Mn@Chloroplast show that its morphology consists of irregularly shaped spheres, and according to... Figure 2 SEM images of EGCG-Mn from the middle chloroplast show that it accumulates on the surface of chloroplasts, which helps chloroplasts overcome stomach acid and better reach intestinal lesions to exert their effects. Figure 2Zeta potential analysis of C showed that the zeta potential of EGCG-Mn@Chloroplast was -18.7, which further proves that the material can exist stably in intestinal fluid.

[0037] Application Example 1: Free radical scavenging and in vitro antioxidant capacity of EGCG-Mn@Chloroplast ABTS, SOD, hydroxyl radicals (•OH), and superoxide anions (O2) - EGCG-Mn@Chloroplast is often used to evaluate the free radical scavenging ability and antioxidant capacity of materials. In this study, we used different concentrations of EGCG-Mn@Chloroplast prepared in Example 1 to explore its scavenging effect on these four antioxidant indicators.

[0038] according to Figure 3 The detection results showed that as the concentration of EGCG-Mn@Chloroplast increased from 0.25 mg / mL to 1 mg / mL, the ABTS clearance efficiency exceeded 80%. Figure 3 (A). For hydroxyl radicals (•OH), the scavenging efficiency approached 80% as the material concentration gradually increased to 0.4 mg / mL. Figure 3 (B), in addition, the material is effective against superoxide anions (O2). - Both ) and SOD also showed good scavenging effects, with scavenging rates exceeding 60% when the material concentration reached 0.4 mg / mL. Figure 3 The four indicators (C and D) all showed a clear dose-dependent effect. These data indicate that EGCG-Mn@Chloroplast possesses strong antioxidant capacity, effectively neutralizing various reactive oxygen species, and its scavenging effect is positively correlated with concentration.

[0039] In addition, the antioxidant capacity of EGCG-Mn@Chloroplast was further verified by electron spin resonance (ESR).

[0040] The results showed that with the addition of EGCG-Mn@Chloroplast, the peak value of the ESR signal of the •DPPH radical decreased significantly. Figure 3 Similarly, hydroxyl radicals (E); Figure 3 (F) and superoxide anion ( Figure 3 The ESR spectral intensity of EGCG-Mn@Chloroplast also decreased significantly, indicating that EGCG-Mn@Chloroplast has excellent free radical scavenging ability and in vitro antioxidant capacity.

[0041] Application Example 2: The restorative effect of EGCG-Mn@Chloroplast on DSS-induced IBD and its alleviating effect on enteritis-related kidney inflammation. 2.1 Treatment of IBD with EGCG-Mn@Chloroplast Laboratory animals: female BALB / c Test substance: EGCG-Mn@Chloroplast Groups: NC, DSS, EGCG-Mn@Chloroplast First, IBD was induced in female BALB / c mice using 3% DSS in free drinking water. After 5 days of modeling, when blood was observed in the mice's feces, the mice were administered medication by gavage. Each mouse in the treatment group was given 0.2 mL of medication per dose, while the NC and DSS groups were given 0.2 mL of physiological saline for 3 consecutive days. The mice were evaluated on day 9. Figure 4 (A). It was found that mice modeled by DSS experienced a significant decrease in body weight, indicating that DSS successfully induced a mouse colitis model. From day 7 onwards, the rate of body weight loss in the treatment group was significantly lower than that in the model group. Figure 4 In addition, the DAI score of mice treated with EGCG-Mn@Chloroplast was significantly lower than that of mice in the DSS model group. Figure 4 (C), which indicates that EGCG-Mn@Chloroplast has an excellent therapeutic effect on DSS-induced colitis in mice.

[0042] 2.2 Mouse colon sections treated with hematoxylin and eosin (H&E) according to Figure 5 The colonic tissue structure of the NC group mice shown in Figure A is intact, the intestinal glands are arranged regularly, and there is no obvious inflammatory cell infiltration, showing a normal colonic tissue morphology. According to... Figure 5 As shown in Figure B, the NC group mice exhibited significant colonic damage, with disruption of the intestinal mucosal structure, extensive infiltration of inflammatory cells, and loss of tissue integrity; while according to Figure 5 As shown in Figure C, the colonic tissue damage in the EGCG-Mn@Chloroplast group mice was significantly repaired, the intestinal gland structure was basically restored to normal, the infiltration of inflammatory cells was significantly reduced, and the tissue structure was consistent with that of the NC group. This indicates that EGCG-Mn@Chloroplast has a significant repair effect in the DSS-induced colonic injury model in mice and can effectively improve the pathological damage of colonic tissue.

[0043] 2.3 Expression of genes related to intestinal barrier integrity and pyroptosis in mice Regarding mRNA, Occludin and E-cadherin are commonly used indicators for assessing intestinal barrier integrity. Mice modeled with DSS show Occludin (… Figure 6 (A) and E-Cadherin ( Figure 6The expression of the intestinal barrier integrity genes in mice treated with EGCG-Mn@Chloroplast was significantly reduced, while the expression of genes related to intestinal barrier integrity was significantly restored. This indicates that EGCG-Mn@Chloroplast has a good post-traumatic repair effect on intestinal barrier integrity. Furthermore, in terms of the expression of inflammation- and pyroptosis-related genes such as IL-1β, NLRP3, Caspase 1, and GSDMD, the expression levels of inflammation and pyroptosis genes in mice treated with EGCG-Mn@Chloroplast were lower than those in the DSS model group. Figure 6 C- Figure 6 (F), which further proves that EGCG-Mn@Chloroplast can effectively regulate the expression of pyroptosis genes and has a good effect on improving inflammatory bowel disease modeled by DSS.

[0044] 2.4 Expression of inflammatory genes in mouse kidneys like Figure 7 As shown in Figure A, the expression of the pyroptosis-related gene GSDMD in the kidneys of mice modeled by DSS was significantly increased compared to the control group, while the expression of GSDMD in the EGCG-Mn@Chloroplast group was significantly decreased compared to the DSS group. This indicates that EGCG-Mn@Chloroplast, while regulating inflammation in the intestine, also has a good alleviating effect on pyroptosis in the kidneys. Furthermore, as... Figure 7 As shown in Figure B, the expression of IL-18 was significantly reduced with the introduction of EGCG-Mn@Chloroplast. The above data can prove that this material also has a certain regulatory effect on inflammation of non-target organs.

[0045] Application Example 3: Regulation of the in vivo microenvironment by EGCG-Mn@Chloroplast 3.1 Expression levels of pro-inflammatory factors and renal function indicators in mouse serum To evaluate the regulatory effect of EGCG-Mn@Chloroplast on renal inflammation, inflammatory factors in mouse serum were detected by ELISA. The results showed that, compared with the normal control group (NC), the serum IL-6 level in the DSS group was significantly increased. Figure 8 (A) This indicates that DSS enteritis triggers systemic inflammation; and after intervention with EGCG-Mn@Chloroplast, IL-6 levels were significantly reduced, indicating that the nanoparticles can effectively inhibit the IL-6-mediated inflammatory cascade. Furthermore, serum TNF-α levels in the DSS group were significantly increased in the NC group, while TNF-α levels decreased significantly after EGCG-Mn@Chloroplast treatment, indicating that it has a potent inhibitory effect on the expression of the pro-inflammatory factor TNF-α. Figure 8 (B)

[0046] In addition, serum IL-1β levels in the DSS group were significantly higher than those in the NC group, and IL-1β levels decreased significantly after EGCG-Mn@Chloroplast intervention. Figure 8 (C) further confirmed that this material can downregulate the release of key pro-inflammatory factors and alleviate systemic inflammatory state.

[0047] Regarding renal function indicators, serum urea nitrogen and creatinine levels in the DSS group were significantly higher than those in the blank control group (NC group), indicating impaired renal excretion and metabolic function; after treatment with EGCG-Mn@Chloroplast, the indicators in both groups were significantly reduced and approached normal levels. Figure 8 D- Figure 8 (E). Combined with the results of serum inflammatory factor detection, it can be seen that EGCG-Mn@Chloroplast can exert a significant therapeutic effect on DSS-induced kidney inflammation by inhibiting systemic inflammatory response and improving renal metabolic function, providing a new potential strategy for intervention of enteritis-related kidney injury.

[0048] 3.2 Immunoflow cytometry results Macrophages are an important component of the immune system. They can engulf foreign substances such as bacteria and viruses, clear senescent cells, and regulate immunity. Macrophages can exhibit different functions and states. M1 macrophages are activated by stimulation from bacteria, viruses, etc., and can release inflammatory factors to attack pathogens. However, overactivation can lead to chronic inflammation. M2 macrophages are induced by damage signals or anti-inflammatory factors and are responsible for inhibiting inflammation, promoting tissue repair, and wound healing.

[0049] like Figure 9 The results showed that, comparing the polarization ratio of macrophages in the spleen of mice, the M1 polarization in the model group reached 16.1%, while the proportion of M1 macrophages in the spleen of mice treated with EGCG-Mn@Chloroplast decreased to 6.13%; and Figure 9 The results showed that the proportion of M2 macrophages in the spleen of mice in the model group was only 17.6%, while the number of M2 macrophages in mice after treatment increased to 31.8%, further demonstrating that EGCG-Mn@Chloroplast has the effect of regulating the immune microenvironment in vivo and reducing inflammatory factors.

[0050] T cells are the core cells of the body's adaptive immune system. Their main function is to precisely identify and eliminate pathogens, such as... Figure 9 C and Figure 9As shown in Figure D, the proportions of cytotoxic T cells (CTL cells) and helper T cells in the treatment group were lower than those in the DSS model group, indicating that EGCG-Mn@Chloroplast has the effect of inhibiting excessive immune response and reducing inflammatory damage. Furthermore... Figure 9 China E- Figure 9 As shown in Figure G, the number of pro-inflammatory cells such as DC cells, Th17 cells, and ILC3 cells in the treatment group was lower than that in the model group, further demonstrating that EGCG-Mn@Chloroplast can effectively regulate the in vivo microenvironment and reduce the inflammatory response.

[0051] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing chloroplast- loaded tea polyphenol self-polymerized nanoparticles, characterized by: The steps are: (1) washing and crushing the spinach, and centrifuging to obtain chloroplasts; (2) adding the epigallocatechin gallate solution to the chloroplast solution, and then adding the manganese sulfate monohydrate solution, and shaking on a shaker to obtain the tea polyphenol self-polymer nanoparticles, named EGCG-Mn@Chloroplast; In step (2), the concentration of the chloroplasts is 2-3 mg / mL; the concentration of the epigallocatechin gallate solution is 80-120 ug / mL; and the concentration of the manganese sulfate monohydrate solution is 80-120 ug / mL.

2. The method of claim 1, wherein: In step (2), the concentration of the chloroplasts is 2.5 mg / mL.

3. The method of claim 1, wherein: In step (2), the concentration of the epigallocatechin gallate solution is 100 ug / mL.

4. The method of claim 1, wherein: In step (2), the concentration of the manganese sulfate monohydrate solution is 100 ug / mL.

5. The method of claim 1, wherein: The volume ratio of the chloroplast solution, the epigallocatechin gallate solution, and the manganese sulfate monohydrate solution is 2:1:

2.

6. The method of claim 1, wherein: In step (2), the temperature for shaking on the shaker is 4℃, the rotation speed is 150-250 rpm, and the time is 2-4 h.

7. The chloroplast-loaded tea polyphenol self-polymer nanoparticles prepared by the preparation method of any one of claims 1-6.

8. The use of the chloroplast-loaded tea polyphenol self-polymer nanoparticles prepared by the preparation method of any one of claims 1-6 in the preparation of a drug for treating inflammatory bowel disease.

9. Use according to claim 8, characterized in that: The inflammatory bowel disease is colitis; and the colitis is sodium dextran sulfate-induced colitis.

10. The use of the chloroplast-loaded tea polyphenol self-polymer nanoparticles prepared by the preparation method of any one of claims 1-6 in the preparation of a drug for treating intestinal inflammatory kidney inflammation.

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