Preparation method and application of berberine and isochlorogenic acid nanomedicine

Abstract

The present application relates to the technical field of pharmaceutical chemistry, in particular to a preparation method and application of a berberine and isovitexin nanomedicine. The application is used in the preparation of a drug for treating diseases related to inflammation mediated by neutrophils. The berberine and isovitexin or the structural analogues of berberine and isovitexin form nanoparticles in a carrier-free form through self-assembly. The berberine and isovitexin nanomedicine has a synergistic anti-inflammatory and antioxidant effect, can efficiently inhibit key inflammatory mediators, realize high-efficiency anti-inflammatory and antioxidant synergy, and significantly improve the effect of preventing and treating acute inflammation.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a method for preparing and applying berberine and isochlorogenic acid nanomedicine. Background Technology

[0002] Inflammation is a rapid defensive response of the body to pathogen invasion, tissue damage, or stress. Controlled inflammatory responses are beneficial for the body's defense against infection, promoting tissue repair, and restoring homeostasis. However, uncontrolled inflammation can lead to various acute and chronic inflammatory diseases, including pneumonia, hepatitis, colitis, and sepsis. Therefore, anti-inflammatory treatment is crucial for the treatment of inflammation-related diseases. Currently, clinical treatment mainly relies on glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and biologics. However, clinical studies have shown that existing anti-inflammatory drugs have serious toxic side effects, such as immunosuppression, gastrointestinal damage, and hepatotoxicity and nephrotoxicity. Biologics may trigger cytokine storms, infections, and malignant tumors, and because they typically act on a single target, they are difficult to precisely regulate complex inflammatory networks. Therefore, there is an urgent need to develop novel, highly effective, low-toxicity, and multi-target treatment strategies.

[0003] The pathological process of acute inflammatory diseases is often accompanied by an overactivated immune response and oxidative stress, leading to the release of large amounts of reactive oxygen species (ROS), forming a vicious cycle of "inflammation-oxidative stress," which can cause multiple organ failure in severe cases. Therefore, improving the inflammatory microenvironment through anti-oxidative stress is a highly promising strategy for the prevention and treatment of inflammation.

[0004] Berberine (BBR), an isoquinoline alkaloid, is the main active ingredient in Coptis chinensis, and its content is highest in Coptis chinensis. Isochlorogenic acid is a polyphenolic compound, a collective term for a group of positional isomers of dicaffeoylquinic acid (diCQA); it usually refers to three types: 3,4-diCQA (isochlorogenic acid B), 3,5-diCQA (isochlorogenic acid A), and 4,5-diCQA (isochlorogenic acid C). All are composed of one molecule of quinic acid and two molecules of caffeic acid linked by an ester bond, differing only in the substitution position of the caffeoyl group on the quinic acid ring. Compared with chlorogenic acid (monocaffeoyl), it has one more phenolic ester structure and twice the number of phenolic hydroxyl groups, resulting in higher biological activity. Berberine and isochlorogenic acid are both naturally derived substances, and existing literature records their anti-inflammatory and antioxidant activities when used alone (for example, berberine reduces inflammation by inhibiting the NF-κB pathway, and isochlorogenic acid exerts its antioxidant effect by scavenging free radicals). However, the poor water solubility and rapid metabolism of these drugs greatly limit their clinical application.

[0005] Chinese patent document CN105753859A discloses a method for preparing a chlorogenic acid-berberine coupling compound and its pharmaceutical uses. This prior art discloses that the chlorogenic acid-berberine coupling compound has a significant lipid-lowering effect.

[0006] Chinese patent document CN120732785A discloses a cytotoxic nanoparticle formulation for treating acute respiratory distress syndrome (ARDS), comprising an active ingredient, a liposomal phospholipid bilayer, and a target. The active ingredient consists of a ROS scavenging drug and an anti-inflammatory drug, wherein the anti-inflammatory drug can be selected from berberine or chlorogenic acid. In this prior art, the lipid-soluble anti-inflammatory drug (such as tanshinone IIA) is encapsulated in the phospholipid bilayer, while the water-soluble ROS scavenging drug is encapsulated in the inner aqueous phase. The release rates of the two types of drugs are difficult to synchronize completely, potentially affecting the synergistic effect of "ROS scavenging + anti-inflammation." Furthermore, it relies on a liposomal delivery system, which may trigger allergic reactions in some patients. In addition, it is designed only for ARDS, and its applicability to other inflammatory lung diseases is unclear. Moreover, the administration method is tracheal infusion, which is not suitable for patients who cannot cooperate with intubation, thus limiting its applicability.

[0007] In addition, although the strategy of using nanotechnology to improve the water solubility of drugs has attracted more and more attention from researchers in recent years, such improvement usually relies on the addition of excipients (such as surfactants or stabilizers), which to some extent increases the complexity of preparation and potential biocompatibility.

[0008] In summary, to address the shortcomings of existing technologies, there is an urgent need to provide a more efficient and safe nanomedicine therapy strategy that combines anti-inflammatory and antioxidant properties. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing berberine and isochlorogenic acid nanomedicine and its application, particularly in the prevention and treatment of acute inflammation.

[0010] One application of the present invention is provided.

[0011] An application of berberine and isochlorogenic acid composite nanoparticles, wherein the application is in the preparation of a drug for treating neutrophil-mediated inflammatory diseases, wherein the berberine and isochlorogenic acid composite nanoparticles are carrier-free nanoparticles formed by the self-assembly of berberine and isochlorogenic acid or structural analogs of berberine and isochlorogenic acid; and the drug is an anti-inflammatory and antioxidant drug.

[0012] Furthermore, the molar ratio of berberine to isochlorogenic acid or the structural analogue of berberine to isochlorogenic acid is 1:1~4.

[0013] In some specific embodiments, the molar ratio of berberine to isochlorogenic acid or the structural analogue of berberine to isochlorogenic acid includes 1:1, 1:2, 1:3 or 1:4.

[0014] In some preferred embodiments, the molar ratio of berberine to isochlorogenic acid or a structural analog of isochlorogenic acid is 1:2.

[0015] Furthermore, the particle size range of the berberine and isochlorogenic acid or the structural analogues of the berberine and isochlorogenic acid is 100-300 nm.

[0016] In some preferred embodiments, the particle size range of the berberine and isochlorogenic acid or the structural analogues of the berberine and isochlorogenic acid is 100-150 nm.

[0017] Furthermore, the polydispersity index of the berberine and isochlorogenic acid or the structural analogue of the berberine and isochlorogenic acid is less than or equal to 0.3.

[0018] In some preferred embodiments, the polydispersity index of the berberine and isochlorogenic acid or the structural analogue of the berberine and isochlorogenic acid is less than 0.3.

[0019] Furthermore, the drug prevents and treats neutrophil-mediated acute inflammation by targeting and inhibiting the NLRP3 inflammasome and the NF-κB pathway, or by downregulating the expression of COX2 and iNOS proteins.

[0020] Furthermore, the neutrophil-mediated inflammatory diseases include pneumonia or hepatitis.

[0021] In some specific embodiments, the neutrophil-mediated inflammatory disease is acute liver failure.

[0022] In some specific embodiments, the neutrophil-mediated inflammatory disease is acute liver injury.

[0023] In some specific embodiments, the neutrophil-mediated inflammatory disease is acute pneumonia.

[0024] In some specific embodiments, the isochlorogenic acid includes isochlorogenic acid B, isochlorogenic acid A, or isochlorogenic acid C; the structural analogues of the isochlorogenic acid include 1,3,5-dicaffeoylquinic acid or 1,4,5-dicaffeoylquinic acid.

[0025] Another invention of the present invention may also provide a preparation method.

[0026] A method for preparing berberine and isochlorogenic acid nanomedicine, wherein the berberine and isochlorogenic acid nanomedicine comprises carrier-free nanoparticles formed by the self-assembly of berberine and isochlorogenic acid or structural analogs of berberine and isochlorogenic acid, and the specific steps include: S1: Dissolve berberine and isochlorogenic acid in a first solvent and adjust the pH to neutral to obtain a first mixed solution; the molar ratio of berberine to isochlorogenic acid is 1:1~4; then slowly add the first mixed solution dropwise to a second solvent, and set the temperature of the second solvent to 50℃-70℃; stir thoroughly to obtain a second mixed solution; The first solvent is an organic solvent, including one or more of dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, or tetrahydrofuran; the second solvent includes ultrapure water, phosphate buffer, or physiological saline. S2: Dialyze the second mixed solution to obtain the berberine and isochlorogenic acid nanomedicine.

[0027] In some specific embodiments, the isochlorogenic acid includes isochlorogenic acid B, isochlorogenic acid A, or isochlorogenic acid C; the structural analogues of the isochlorogenic acid include 1,3,5-dicaffeoylquinic acid or 1,4,5-dicaffeoylquinic acid.

[0028] In some specific embodiments, in S2, a dialysis bag is used to dialyze with ultrapure water for 12-48 hours, and the ultrapure water is changed every 4 hours. The molecular weight cutoff of the dialysis bag is 1000Da-3000Da.

[0029] In some specific embodiments, in S1, a microfluidic device is used to introduce the first mixed solution and the second solvent into a Y-type mixer or a spiral mixing channel to mix them and obtain a second mixed solution; the flow rate ratio of the first mixed solution to the second solvent in the Y-type mixer or spiral mixing channel is 5-20:1, and the flow rate is 1-20 mL / h.

[0030] Another aspect of the present invention may provide a pharmaceutical preparation.

[0031] A drug for treating a neutrophil-mediated inflammatory disease, the drug comprising carrier-free nanoparticles formed by the self-assembly of berberine and isochlorogenic acid or a structural analog of berberine and isochlorogenic acid; wherein the molar ratio of berberine to isochlorogenic acid or the structural analog of berberine to isochlorogenic acid is 1:1 to 4.

[0032] Furthermore, the drug is an anti-inflammatory and antioxidant drug.

[0033] In some specific embodiments, the neutrophil-mediated inflammatory disease is acute liver failure.

[0034] In some specific embodiments, the neutrophil-mediated inflammatory disease is acute liver injury.

[0035] In some specific embodiments, the neutrophil-mediated inflammatory disease is acute pneumonia.

[0036] Beneficial technical effects: (1) This invention provides a carrier-free composite nanoparticle formed by the self-assembly of berberine and isochlorogenic acid or structural analogs of berberine and isochlorogenic acid. In some specific pharmaceutical applications, this composite nanoparticle can be prepared as BBR-ICAB nanomedicines. This nanomaterial is formed by the direct formation of a stable nanostructure by berberine and isochlorogenic acid or structural analogs of isochlorogenic acid through intermolecular forces (such as hydrogen bonds or π-π stacking), without the need for any exogenous excipients or carriers. The preparation process is simple and efficient (reducing production steps and raw material costs), and the resulting nanomedicine has high safety (reducing the risk of potential impurities). The preparation method provided by this invention optimizes key processes such as solvent selection and material ratio to screen nanoparticles with suitable particle size and good uniformity and stability for clinical treatment.

[0037] (2) The carrier-free composite nanoparticles provided by this invention, as a drug, can efficiently inhibit key inflammatory mediators (such as TNF-α and IL-6) and oxidative stress markers, and have been shown to have better efficacy than single-component drugs. Furthermore, by inhibiting the release of pro-inflammatory cytokines and clearing ROS oxidation, it effectively breaks the vicious cycle of "inflammation-oxidative stress," providing a novel strategy for the treatment of acute inflammation. For various acute inflammatory diseases, this drug targets and inhibits the NLRP3 inflammasome and NF-κB pathways, while downregulating COX2 and iNOS protein expression, and simultaneously regulates common pathways to exert therapeutic effects, thereby preventing and treating neutrophil-mediated acute inflammation, thus providing a universal platform. At the same time, its structural analogue design allows for further optimization of pharmacodynamics and pharmacokinetics.

[0038] (3) Compared with traditional anti-inflammatory drugs, this drug, through multi-target synergistic action, not only significantly reduces toxic side effects but also avoids risks such as immunosuppression, gastrointestinal damage, and hepatotoxicity and nephrotoxicity. Simultaneously, the carrier-free nanostructure enhances drug stability and bioavailability, ensuring precise accumulation and long-lasting release at the site of inflammation. Experimental data show that this drug exhibits significant anti-inflammatory and antioxidant activity in mouse models of pneumonia and hepatitis, demonstrating broad prospects for clinical application. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0040] Figure 1 The particle size distribution of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention is shown. Figure 2 This is a transmission electron microscope image of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention; Figure 3 This invention relates to the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB). 1 H NMR spectrum; Figure 4 This is a stability analysis diagram of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention; Figure 5 This is a molecular dynamics analysis diagram of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention; wherein (A) is a diagram of the structural changes of the BBR-ICAB system at different time points during the simulation, (B) is a diagram of the structural changes of the BBR-ICAB system in the initial and final states of the simulation, and (C) is a diagram of the intermolecular interaction mode of the BBR-ICAB system. Figure 6 This is a graph showing the in vitro anti-inflammatory effects of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention compared with other control groups; where (a) is the relative expression level of TNF-α in different control groups, (b) is the relative expression level of IL-1β in different control groups, (c) is the relative expression level of IL-6 in different control groups, and (d) is the relative expression level of IL-8 in different control groups. Figure 7 This is a graph showing the in vitro antioxidant effect of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention. Figure 8 This is an analytical diagram showing the effect of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention on inhibiting the production of reactive oxygen species in neutrophils stimulated by PMA, compared with other control groups. Figure 9 This is a graph showing the anti-inflammatory effects of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention and other control groups in acute pneumonia; where (a) is the relative expression level of TNF-α in different control groups, (b) is the relative expression level of IL-6 in different control groups, (c) is the relative expression level of IL-8 in different control groups, and (d) is the relative expression level of MPO in different control groups. Figure 10This is a graph showing the antioxidant effects of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention compared with other control groups in acute pneumonia. Figure 11 This is a graph showing the liver function improvement effect of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention compared with other control groups in the treatment of acute hepatitis; where (a) is the ALT content of different test groups, (b) is the AST content of different test groups, (c) is the LDH content of different test groups, and (d) is the ALP content of different test groups. Figure 12 This is a graph showing the effect of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention on inhibiting liver inflammation in mice with acute liver injury compared with other control groups; where (a) is the relative expression level of IL-6 in different test groups, (b) is the relative expression level of IL-8 in different test groups, and (c) is the relative expression level of MPO in different test groups. Figure 13 This is a graph showing the effect of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention on inhibiting oxidative stress in the liver of mice with acute liver injury compared with other control groups. Figure 14 The graph shows the effect of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention on reversing liver function in mice with acute liver failure compared with other control groups; where (a) is the ALT content of different test groups and (b) is the AST content of different test groups. Figure 15 This is a graph showing the effect of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention on inhibiting hepatocyte apoptosis in mice with acute liver failure compared with other control groups. Figure 16 A protein quantification diagram for analyzing the anti-inflammatory mechanism of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention; Figure 17 This is a protein quantification diagram for analyzing the antioxidant mechanism of the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention; Figure 18 This is an analysis diagram of the inhibition of the NLRP3 / ASC signaling pathway by the berberine-isochlorogenic acid B self-assembled excipient-free nanomedicine (BBR-ICAB) of the present invention; wherein (a) is the relative expression level of NLRP3 in different detection groups, (b) is the relative expression level of ASC in different detection groups, and (c) is the relative expression level of IL-18 in different detection groups. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0043] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0044] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, even more typically + / -0.5%.

[0045] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example: Application of the berberine and isochlorogenic acid nanomedicine, wherein the application is to prepare a drug for preventing and treating neutrophil-mediated acute inflammation; the drug contains a carrier-free nanomedicine formed by the self-assembly of berberine and isochlorogenic acid or a structural analog of isochlorogenic acid. The molar ratio of berberine to isochlorogenic acid or a structural analog of isochlorogenic acid is 1:(0.3~4).

[0049] In some specific embodiments, the molar ratio of berberine to isochlorogenic acid or a structural analog of isochlorogenic acid is one of 1:1, 1:2, 1:3, 1:4, 2:1, or 3:1.

[0050] In some specific embodiments, the molar ratio of berberine to isochlorogenic acid or a structural analog of isochlorogenic acid is 1:2.

[0051] In some specific embodiments, the drug is an anti-inflammatory and antioxidant drug that prevents and treats neutrophil-mediated acute inflammation by targeting and inhibiting the NLRP3 inflammasome and NF-κB pathway, while downregulating the expression of COX2 and iNOS proteins; the particle size of the drug is 100-150 nm.

[0052] In some specific embodiments, the neutrophil-mediated inflammatory diseases include pneumonia, hepatitis, colitis, and sepsis.

[0053] In some specific embodiments, the isochlorogenic acid includes one or more of isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C; the structural analogues of the isochlorogenic acid include one or a mixture of two of 1,3,5-dicaffeoylquinic acid and 1,4,5-dicaffeoylquinic acid.

[0054] In some specific embodiments, the isochlorogenic acid is isochlorogenic acid B (ICAB). The self-assembled structure of berberine and isochlorogenic acid B nanoparticles is maintained by hydrogen bonding and π-π conjugation.

[0055] The structural formula of isochlorogenic acid B is: ; The structural formula of chlorogenic acid is: .

[0056] This embodiment provides a method for preparing nanomedicines of berberine and isochlorogenic acid or isochlorogenic acid analogs, the specific steps of which are as follows: S1: Berberine and isochlorogenic acid or a structural analog of isochlorogenic acid are dissolved in a first solvent and the pH is adjusted to neutral to obtain a first mixed solution; then the first mixed solution is mixed with a second solvent to obtain a second mixed solution; in step S1, the first solvent is an organic solvent, including one or more of dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol or tetrahydrofuran; the second solvent is ultrapure water, phosphate buffer or physiological saline.

[0057] S2: Dialyze the second mixed solution from step S1 to obtain self-assembled nanoparticles; In step S2, a dialysis bag is used to dialyze with ultrapure water for 12 hours, and the ultrapure water is changed every 4 hours. The molecular weight cutoff of the dialysis bag is 2000 Da.

[0058] S3: Collect self-assembled nanoparticles, freeze-dry them under vacuum to obtain carrier-free nanomedicines.

[0059] In some specific embodiments, the mixing of the first mixed solution and the second solvent in step S1 is to add the first mixed solution dropwise into the second solvent and stir at a constant temperature to obtain the second mixed solution; Alternatively, a microfluidic instrument can be used to introduce the first mixed solution and the second solvent into a Y-type mixer or a spiral mixing channel to mix them and obtain a second mixed solution.

[0060] In some specific embodiments, the first mixed solution is slowly added dropwise to 60°C ultrapure water, phosphate buffer, or physiological saline, and stirred at a constant temperature for 30 minutes to obtain the second mixed solution.

[0061] In some specific embodiments, the flow rate ratio of the first mixed solution to the second solvent in the Y-type mixer or spiral mixing channel is (5-20):1, and the total flow rate is 1-20 mL / h.

[0062] The technical solution of the present invention will be further described in detail below through several specific embodiments.

[0063] Specific Example 1: The molar ratio of berberine (BBR)-isochlorogenic acid B (ICAB) excipient-free nanomedicine (BBR-ICAB) was investigated using isochlorogenic acid B.

[0064] The preparation method of this berberine-isochlorogenic acid B nanomedicine includes the following specific steps: S1: Weigh berberine and isochlorogenic acid B in different molar ratios and dissolve them in the first solvent (a mixture of DMSO and methanol). The amount of the first solvent added is 2 mg / 250 μL. Adjust the pH to neutral to obtain the first mixed solution. Then, slowly add the first mixed solution dropwise to the second solvent, ultrapure water at 60℃ and stir at a constant temperature for 30 min to obtain the mixed solution. S2: Place the mixed solution from step S1 into a dialysis bag (molecular weight cutoff: 2000 Da) and dialyze with ultrapure water for 12 h to remove unassembled berberine and isochlorogenic acid B, as well as DMSO and methanol. Change the water every 4 h to obtain self-assembled berberine-isochlorogenic acid B nanoparticles. S3: Collect the self-assembled nanoparticles inside the dialysis bag, freeze-dry them under vacuum to obtain carrier-free berberine-isochlorogenic acid B nanomedicine.

[0065] Berberine-isochlorogenic acid B nanomedicines (also known as BBR-ICAB nanomedicines) prepared with different molar ratios were characterized, and the results are shown in Table 1.

[0066] Table 1. Average particle size, PDI, and zeta potential values ​​of self-assembled nanomedicines of berberine and isochlorogenic acid B at different molar ratios. Table 1 shows that the molar ratio of berberine (BBR) to isochlorogenic acid B (ICAB) affected the average hydrated particle size, polydispersity index (PDI), and zeta potential of the chlorogenic acid-berberine nanodrug. When the molar ratio of berberine to isochlorogenic acid B changed from 1:0.5 to 1:4, the average hydrated particle size, PDI, and zeta potential of the BBR-ICAB nanodrug varied within the ranges of 213.13±56.37 to 403.33±59.56 nm, 0.269±0.02 to 0.68±0.03, and -30.77±4.02 mV to -32.27±1.55, respectively. Compared with other ratios, the BBR-ICAB nanodrug prepared at a molar ratio of 1:2 had the smallest average hydrated particle size (105.87±3.61 nm) and a narrower particle size distribution. Figure 1 The PDI of the BBR-ICAB carrier-free nanoparticles was 0.269±0.02, and the zeta potential was -30.77±0.12 mV. PDI is one of the indicators for evaluating the uniformity and stability of nanomedicines. A PDI of 0.3 or less indicates uniform distribution of the nanomedicine, while a small particle size ensures the stable existence of the carrier-free BBR-ICAB nanomedicine in the blood. The zeta potential reflects the degree of surface charge of the particles and their interaction with ions in the surrounding liquid; a higher absolute value of the zeta potential indicates better system stability. Therefore, a molar ratio of berberine to isochlorogenic acid B of 1:2 was chosen as the optimal ratio for preparing the carrier-free BBR-ICAB nanomedicine. Its morphology is as follows: Figure 2 As shown, from Figure 2 As can be seen, the BBR-ICAB carrier-free nanomedicine has a uniform particle distribution and a particle size of approximately 105.9 ± 3.6 nm.

[0067] like Figure 3 The image shows BBR-ICAB nanomedicine. 1¹H NMR spectra were obtained using an Agilent DD2 NMR spectrometer. The ¹H NMR frequency was 400 MHz, and the ¹³C NMR frequency was 100 MHz. The test temperature was 25 °C. Deuterated DMSO was used as the test solvent, and calibration was performed using residual peaks from the deuterated DMSO. Standard parameters in the field were used. The obtained ¹H NMR spectrum of BBR-ICAB showed major proton signals simultaneously attributed to BBR and ICAB, indicating molecular integration. The characteristic chemical shifts of BBR appeared at 9.87, 8.92, 8.18, 7.98, 7.76, 7.06, 6.14, 4.95, 4.04, and 3.36 ppm, while the characteristic shifts of ICAB appeared at 9.57, 9.12, 7.42, 7.00, 6.93, 6.71, 6.16, 5.38, 4.90, 4.33, 4.05, and 3.33 ppm. It is worth noting that in the BBR-ICAB spectrum, some proton signals of ICAB are broadened or not completely separated, which may be due to the formation of large supramolecular assemblies by strong π-π stacking and hydrogen bonding.

[0068] Using the above preparation method, the first solvent was replaced to prepare berberine (BBR)-isochlorogenic acid B (ICAB) excipient-free nanomedicine (BBR-ICAB). The influence of the choice of the first solvent in the preparation process on the performance of the prepared BBR-ICAB self-assembled nanoparticles was investigated, as shown in Table 2.

[0069] Table 2 Performance analysis of BBR-ICAB self-assembled nanoparticles prepared with different solvents Table 2 shows that when dimethyl sulfoxide (DMSO) and methanol were used as the first solvent, the prepared BBR-ICAB self-assembled nanoparticles had the lowest PDI value (0.269±0.02) and a relatively high absolute value of zeta potential (-30.77 mV), indicating that the nanoparticles had good uniformity and stability under this solvent combination. While N,N-dimethylformamide could also form self-assembled nanoparticles as a solvent, the PDI value increased (0.355±0.007) and the absolute value of zeta potential decreased (-27.98±5.73 mV), indicating that its uniformity and stability were slightly inferior to the combination of DMSO and methanol. When ethanol and tetrahydrofuran were used alone as solvents, stable BBR-ICAB self-assembled nanoparticles were not successfully prepared; aggregation occurred after a period of time. This may be because the interaction between these two solvents and berberine and isochlorogenic acid B was weak, failing to effectively promote the self-assembly process. In Comparative Group 1, although methanol was used as the first solvent, self-assembled nanoparticles could still be formed, but the PDI value and absolute value of the zeta potential were not as good as those of the combination of dimethyl sulfoxide and methanol. In Comparative Group 2, when dimethyl sulfoxide was used as the first solvent, the BBR-ICAB self-assembled nanoparticles exhibited a smaller PDI value (0.287±0.031) and a relatively higher absolute value of the zeta potential (-29.57mV) upon completion of preparation, but agglomeration occurred after a period of time, rendering them unstable. Therefore, considering the uniformity, stability, and feasibility of the preparation process, a mixture of dimethyl sulfoxide and methanol is preferred as the first solvent for preparing BBR-ICAB carrier-free nanomedicines.

[0070] Specific Example 2: Stability analysis of BBR-ICAB self-assembled nanoparticles, specifically as follows: The BBR-ICAB self-assembled nanoparticles (Group 3) prepared in Example 1 with a molar ratio of berberine to isochlorogenic acid B of 1:2 were distributed at a concentration of 1 mg / mL in phosphate-buffered saline (PBS) or DMEM medium (FBS) containing 20% ​​fetal bovine serum. The average particle size was measured after incubation at 4°C or 37°C for 0-7 days. Results are as follows... Figure 4 As shown, the BBR-ICAB self-assembled nanoparticles did not exhibit significant changes in particle size after 7 days of incubation in PBS or FBS, indicating good stability.

[0071] Specific Example 3: A healthy control group (Control), an animal model (Model), a BBR group (single component), an ICAB group (single component), and a BBR-ICAB nanoparticle treatment group (BBR-ICAB) were set up respectively. Molecular dynamics analysis was performed on the self-assembly mechanism of BBR-ICAB nanoparticles, specifically as follows: All-atom molecular dynamics simulations were performed on the BBR-ICAB self-assembled nanoparticles prepared in group 3 of specific Example 1 using the GROMACS software package. OPLSAA force fields were used to describe the molecules, and Clions were used to neutralize the system charge to construct the simulated system. The results were visualized using PyMOL-3.0.3. Figure 5 As shown. From Figure 5 As can be seen, BBR and ICAB form a stable self-assembled structure through hydrogen bonding and π-π stacking (conjugation) between aromatic rings.

[0072] Specific Example 4: Verification of the in vitro anti-inflammatory effect of BBR-ICAB self-assembled nanoparticles, specifically as follows: RAW264.7 macrophages were seeded in 6-well plates and cultured overnight. Afterward, the cells were stimulated with 500 ng / mL LPS. After incubation for 12 hours with BBR (12.5 μM), ICAB (25 μM), or BBR-ICAB self-assembled nanoparticles (25 μM, ICAB) prepared in Group 3 of Example 1, the protein expression levels of TNF-α, IL-1β, IL-6, and IL-8 in the cell supernatant were measured by ELISA. Pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8 significantly increase during acute inflammation, activating immune cells to mediate inflammatory responses. Overexpression of cytokines can lead to local or systemic inflammatory responses, and their levels are important indicators for assessing the degree of infection and for early warning of cytokine storms. Figure 6 As shown, Figure 6 In the figure, (a) represents the relative expression level of TNF-α in different detection groups. Figure 6 (b) in the figure represents the relative expression levels of IL-1β in different detection groups. Figure 6 In the figure, (c) represents the relative expression level of IL-6 in different detection groups. Figure 6 (d) represents the relative expression level of IL-8 in different detection groups. The results showed that BBR-ICAB significantly reduced the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 in RAW264.7 cells stimulated by LPS, and the effect was better than that of single drugs (BBR or ICAB), confirming that it has a good anti-inflammatory effect.

[0073] Specific Example 5: Verification of the in vitro antioxidant effect of BBR-ICAB self-assembled nanoparticles. A healthy control group (Control), an animal model (Model), a BBR group (single component), an ICAB group (single component), and a BBR-ICAB nanoparticle treatment group (BBR-ICAB) were set up. Hydroxyl radicals (…) were used… The OH group was removed to verify this. Specifically: The BBR-ICAB self-assembled nanoparticles prepared in Group 3 of Specific Example 1 were diluted to 0 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively, and then incubated with hydroxyl radicals (·OH) and the ·OH concentration was tested using a hydroxyl radical kit.

[0074] Excessive production of hydroxyl radicals (·OH) during acute inflammation indiscriminately attacks DNA, proteins, and lipids, leading to cell death, disrupting cell membrane integrity, and causing leakage of cell contents. It can also inactivate the antiprotease system, causing uncontrolled protease activity and subsequently damaging the extracellular matrix, including elastic fibers and collagen fibers. ·OH can also directly damage vascular endothelial cells, leading to increased vascular permeability and causing tissue edema and exudation. Figure 7 As shown, BBR-ICAB self-assembled nanoparticles can significantly scavenge ·OH, confirming their excellent antioxidant capacity.

[0075] Simultaneously, the in vitro antioxidant effect of BBR-ICAB self-assembled nanoparticles in inhibiting the production of reactive oxygen species in neutrophils stimulated by phorbol ester (PMA) was verified. Figure 8 The figure shown is a graph comparing the single-component performance of BBR-ICAB nanomedicine with that of the control group in inhibiting PMA-stimulated reactive oxygen species production in neutrophils. Figure 8 The fluorescence intensity of different detection groups was analyzed, and compared with the healthy control group (Control), animal model (Model), BBR group (single component), and ICAB group (single component), it was confirmed that BBR-ICAB self-assembled nanomedicine can inhibit the production of reactive oxygen species in neutrophils caused by PMA stimulation. The antioxidant effect of BBR-ICAB self-assembled nanomedicine is higher than that of single component BBR or ICAB.

[0076] Specific Example 6: Verification of the anti-inflammatory effect of BBR-ICAB self-assembled nanoparticles in an acute pneumonia model, specifically as follows: After BALB / c mice were induced to develop a model using LPS, they were injected with physiological saline (Model group), BBR (5 mg / kg) (BBR group), ICAB (15 mg / kg) (ICAB group), and BBR-ICAB self-assembled nanoparticles (BBR at 5 mg / kg) prepared in group 3 of specific example 1 (BBR-ICAB group). After 12 h, bronchoalveolar lavage fluid was collected, cells were collected by centrifugation, and the mRNA levels of TNF-α, IL-6, IL-8, and MPO were measured by RT-qPCR.

[0077] In a mouse model of acute lung injury, experimental results (such as...) Figure 9 As shown in the figure, Figure 9 (a) represents the relative expression level of TNF-α in the alveoli of mice in different groups. Figure 9 (b) in the figure represents the relative levels of IL-6 in the alveoli of mice in different groups. Figure 9 (c) represents the relative expression level of IL-8 in the alveoli of mice in different groups. Figure 9 (d) represents the relative expression level of MPO in the alveoli of mice in different groups. Compared with the healthy control group (Control), animal model (Model), BBR group (single component), and ICAB group (single component), the BBR-ICAB self-assembled nanoparticle treatment group exhibited significant anti-inflammatory protective effects. The BBR-ICAB self-assembled nanoparticles significantly reduced the mRNA expression levels of TNF-α, IL-6, IL-8, and MPO in the LPS-induced mouse acute lung injury model. This result is consistent with the in vitro anti-inflammatory efficacy verification experiment, further confirming the good anti-inflammatory effect of BBR-ICAB self-assembled nanoparticles in animal models. Furthermore, the relative expression level of MPO (myeloperoxidase), a marker of neutrophil activation, was also significantly reduced in the BBR-ICAB self-assembled nanoparticle treatment group, indicating that the nanoparticles can effectively inhibit neutrophil activation, thereby alleviating the inflammatory response during acute lung injury.

[0078] In summary, BBR-ICAB self-assembled nanoparticles effectively inhibited the excessive activation of bronchial epithelial cells, specifically by significantly downregulating the expression levels of key pro-inflammatory factors secreted by these cells, including TNF-α, IL-6, IL-8, and MPO. This finding confirms that BBR-ICAB can effectively block the inflammatory cascade and reduce the infiltration and damage of inflammatory cells to lung tissue.

[0079] Specific Example 7: Verification of the antioxidant effect of BBR-ICAB self-assembled nanoparticles in an acute pneumonia model, specifically as follows: Modeling and drug administration were performed according to the method described in Specific Example 6. Bronchoalveolar lavage fluid was collected 12 hours later, cells were collected by centrifugation, and the residual hydrogen peroxide content was detected using a hydrogen peroxide detection kit. Figure 10As shown, compared with the healthy control group (Control), the residual hydrogen peroxide content in the animal model group (Model) was significantly increased, indicating that the ALI model was successfully constructed and that there was significant oxidative stress damage. Compared with the animal model group (Model), the BBR group (single component), and the ICAB group (single component), the residual hydrogen peroxide content in the BBR-ICAB group was significantly reduced, revealing that the BBR-ICAB self-assembled nanoparticles have significant antioxidant effects, effectively clearing oxidative stress products and reducing oxidative damage. Combined with the detection results of TNF-α, IL-6, IL-8, and MPO, it indicates that the BBR-ICAB self-assembled nanoparticles can effectively inhibit the release of pulmonary inflammatory factors and the recruitment of neutrophils, thereby reducing the oxidative stress response induced by immune cell infiltration and reducing the production of oxidative products such as hydrogen peroxide. This further corroborates the role of BBR-ICAB self-assembled nanoparticles in improving the pulmonary inflammatory microenvironment and reducing secondary lung tissue damage.

[0080] Specific Example 8: Verification of the liver function improvement effect of BBR-ICAB self-assembled nanoparticles in an acute liver injury model, specifically as follows: After BALB / c mice were induced to develop acute hepatitis using carbon tetrachloride, they were injected with physiological saline (Model group), BBR (5 mg / kg) (BBR group), ICAB (15 mg / kg) (ICAB group), and BBR-ICAB self-assembled nanoparticles (BBR at 5 mg / kg) prepared in group 3 of specific example 1 (BBR-ICAB group). After 24 hours, the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) levels of the mice were measured.

[0081] like Figure 11 As shown, Figure 11 (a) in the table represents serum alanine aminotransferase (ALT) in different groups. Figure 11 (b) in the figure represents the serum aspartate aminotransferase (AST) content in different groups; Figure 11 (c) represents serum lactate dehydrogenase (LDH) in different groups. Figure 11 In the figure, (d) represents the serum alkaline phosphatase (ALP) levels in different groups. In a mouse model of acute hepatitis, comparisons were made with a healthy control group, an animal model, a BBR group (single component), and an ICAB group (single component). The results clearly showed that BBR-ICAB self-assembled nanoparticles significantly reduced serum ALT, AST, ALP, and LDH levels. Carbon tetrachloride, after being metabolized in the liver, produces a large number of free radicals (ROS), which can induce acute liver injury and inflammatory responses. Figure 11The results showed that BBR-ICAB self-assembled nanoparticles had significant hepatoprotective and enzyme-lowering effects, and could significantly improve liver function in model mice.

[0082] Specific Example 9: Analysis of inflammation and antioxidant stress in the liver region of BBR-ICAB self-assembled nanoparticles in an acute liver injury model. Oxidative stress is one of the important mechanisms leading to hepatocyte damage and death during acute liver injury. Specifically: The model was established and drugs were administered according to the method described in Specific Example 8. Mice were sacrificed 24 hours later, and fresh liver tissue was collected. After grinding and pulverizing, total RNA was extracted from some cells using an RNA extraction kit. The mRNA expression levels of IL-6, IL-8, and MPO in the mouse liver tissue were quantitatively detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The hydrogen peroxide content in the remaining cells was detected using a hydrogen peroxide detection kit. Figure 12 and Figure 13 As shown, where Figure 12 In the figure, (a) represents the relative expression level of IL-6 in mouse liver tissue. Figure 12 (b) in the figure represents the relative expression level of IL-8 in mouse liver tissue. Figure 12 (c) represents the relative expression level of MPO in mouse liver tissue. Figure 13 The hydrogen peroxide content in mouse liver tissue was determined for analysis of oxidative stress in mouse liver. Comparisons with a healthy control group, an animal model, a BBR group (single component), and an ICAB group (single component) showed that in a mouse model of acute hepatitis, BBR-ICAB significantly downregulated the mRNA expression levels of IL-6, IL-8, and MPO in liver tissue, inhibiting the inflammatory response in the liver. Figure 13 The results of oxidative stress analysis in the liver tissue of mice with acute liver injury in different groups are shown. Berberine and isochlorogenic acid B in BBR-ICAB self-assembled nanoparticles both exhibit antioxidant activity, capable of scavenging free radicals and inhibiting lipid peroxidation, thereby protecting hepatocytes from oxidative stress damage. Furthermore, treatment with BBR-ICAB significantly reduced the production of reactive oxygen species in liver tissue. This conclusion is consistent with the antioxidant effects demonstrated by BBR-ICAB self-assembled nanoparticles in an acute pneumonia model, further confirming its potential application value in the treatment of acute liver injury. Simultaneously, BBR-ICAB self-assembled nanoparticles provide a new treatment option for acute liver injury through a dual mechanism of anti-inflammation and anti-oxidation.

[0083] Specific Example 10: Verification of the liver function improvement effect of BBR-ICAB self-assembled nanoparticles in a mouse model of acute liver failure, specifically as follows: After inducing acute liver failure in BALB / c mice through a single intraperitoneal injection of carbon tetrachloride, the mice were injected with saline (model group), BBR (5 mg / kg) (BBR group), ICAB (15 mg / kg) (ICAB group), and BBR-ICAB self-assembled nanoparticles (BBR at 5 mg / kg) prepared in group 3 of specific example 1 (BBR-ICAB group). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured 24 hours later. Figure 14 As shown, Figure 14 In the figure, (a) represents the ALT content of different test groups. Figure 14 (b) represents the AST content in different detection groups. Simultaneously, fresh liver tissue was obtained after euthanizing the mice. The tissue was dehydrated, fixed, paraffin-embedded, and sectioned according to procedure. TUNEL staining (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique) was performed on the tissue sections after fixation and dehydration using methods commonly used by those skilled in the art to detect hepatocyte apoptosis (e.g., ...). Figure 15 (As shown). Compared with healthy mice (Control), ALF mice in the animal model group (Model) showed elevated liver index and significantly increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. From Figure 14 A comparison of the BBR group, ICAB group, and BBR-ICAB group showed that after treatment with BBR, ICAB, and BBR-ICAB, these typical pathological changes were significantly reversed, and the efficacy of the BBR-ICAB group was significantly better than that of free BBR or ICAB alone. Meanwhile, as... Figure 15 As shown, Figure 15 The scale bars were the same for all test groups. The TUNEL staining results of different test groups showed that BBR-ICAB treatment could significantly inhibit liver cell apoptosis.

[0084] Specific Example 11: Analysis of the anti-inflammatory mechanism of BBR-ICAB self-assembled nanoparticles, specifically as follows: RAW264.7 macrophages were seeded in 6-well plates and cultured overnight. After incubation, the cells were stimulated with 500 ng / mL LPS. After incubation for 12 h with BBR (12.5 μM), ICAB (25 μM), or BBR-ICAB self-assembled nanoparticles (25 μM, ICAB) prepared in Group 3 of Example 1, a portion of the cells were collected for protein extraction and Western blot analysis. Total RNA was extracted from another portion of the cells using an RNA extraction kit, and the mRNA expression levels of NLRP3, ASC, and IL-18 were detected by RT-qPCR. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control in protein experiments to analyze the expression levels of the core proteins of the inflammasome signaling pathway: NLRP3, P-NF-κb, ASC, COX2, and iNOS.

[0085] like Figure 16 The image shows the analysis of the anti-inflammatory mechanism of BBR-ICAB self-assembled nanoparticles, such as... Figure 17 The antioxidant mechanism analysis of BBR-ICAB self-assembled nanoparticles is shown. Comparisons with a healthy control group (Control), an animal model (Model), a BBR group (single component), and an ICAB group (single component) revealed that BBR-ICAB self-assembled nanoparticles can target and inhibit the NLRP3 inflammasome and the NF-κB pathway. LPS-induced macrophages showed significantly increased expression levels of iNOS and COX2. However, BBR-ICAB treatment significantly inhibited these elevated iNOS and COX2 expressions, indicating that the iNOS / COX2 inflammatory pathway was blocked. Notably, subsequent studies found that BBR treatment alone did not significantly alter iNOS and COX-2 expression, suggesting that the inhibitory effect of BBR-ICAB nanomedicines primarily originates from its ICAB component. BBR-ICAB self-assembled nanoparticles can target and inhibit the NLRP3 inflammasome and NF-κB pathway, while downregulating the expression of proteins such as COX2 and iNOS. Therefore, BBR-ICAB self-assembled nanoparticles effectively curb the inflammatory cascade by combining anti-inflammatory and antioxidant effects.

[0086] like Figure 18 As shown, total RNA was extracted from a subset of cells using an RNA extraction kit. The mRNA expression of inflammatory cytokines NLRP3, ASC, and IL-18 in the cells was then quantitatively detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR) to investigate the inhibition of the NLRP3 / ASC signaling pathway by BBR-ICAB nanomedicine. Figure 18 In the table, (a) represents the relative expression level of NLRP3. Figure 18 In the table, (b) represents the relative expression level of ASC. Figure 18(c) represents the relative expression level of IL-18. RT-qPCR results showed that LPS stimulation significantly increased the mRNA expression levels of NLRP3, ASC, and IL-18 compared to the healthy control group (Control). After treatment with BBR and BBR-ICAB, the mRNA levels of NLRP3, ASC, and IL-18 all decreased significantly. Notably, ICAB alone did not alter the mRNA expression of these target genes, indicating that the anti-inflammatory effect of BBR-ICAB is mainly achieved through BBR regulation of the NLRP3 pathway.

[0087] In summary, the berberine-isochlorogenic acid or isochlorogenic acid structural analogues self-assembled carrier-free nanomedicines prepared in this application increase the drug surface area and cell penetration ability, producing a synergistic effect. This combination can target and inhibit inflammation-related signaling pathways, downregulate the expression of inflammatory response and oxidative stress-related proteins, and efficiently inhibit key inflammatory mediators (such as TNF-α and IL-6) and oxidative stress markers, thereby reducing free radical generation and inhibiting the inflammatory cascade reaction, with therapeutic effects superior to single components. The berberine-isochlorogenic acid or isochlorogenic acid structural analogue drug combination prepared in this invention has synergistic anti-inflammatory and antioxidant effects, and has a powerful therapeutic effect on inflammatory diseases such as acute pneumonia and acute liver injury.

[0088] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0089] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An application of berberine and isochlorogenic acid composite nanoparticles, characterized in that, The application is in the preparation of a drug for treating neutrophil-mediated inflammatory diseases, wherein the berberine and isochlorogenic acid composite nanoparticles are carrier-free nanoparticles formed by the self-assembly of structural analogs of berberine and isochlorogenic acid or berberine and isochlorogenic acid; the drug is an anti-inflammatory and antioxidant drug.

2. The application of the berberine and isochlorogenic acid composite nanoparticles according to claim 1, characterized in that, The molar ratio of berberine to isochlorogenic acid or the structural analog of berberine to isochlorogenic acid is 1:1 to 4.

3. The application of the berberine and isochlorogenic acid composite nanoparticles according to claim 1, characterized in that, The particle size range of the berberine-isochlorogenic acid composite nanoparticles is 100-300 nm; or, the polydispersity index of the berberine-isochlorogenic acid composite nanoparticles is less than or equal to 0.

3.

4. The application of the berberine and isochlorogenic acid composite nanoparticles according to claim 1, characterized in that, The drug inhibits the NLRP3 inflammasome and the NF-κB pathway by targeting and inhibiting them; or, the drug simultaneously downregulates the expression of COX2 and iNOS proteins.

5. The application of the berberine and isochlorogenic acid composite nanomedicine according to claim 1, characterized in that, Neutrophil-mediated inflammatory diseases include pneumonia or hepatitis.

6. The application of the berberine and isochlorogenic acid composite nanoparticles according to claim 1, characterized in that, The isochlorogenic acid includes isochlorogenic acid B, isochlorogenic acid A, or isochlorogenic acid C; the structural analogues of the isochlorogenic acid include 1,3,5-dicaffeoylquinic acid or 1,4,5-dicaffeoylquinic acid.

7. A method for preparing berberine and isochlorogenic acid nanomedicine, characterized in that, The berberine and isochlorogenic acid nanomedicine comprises carrier-free nanoparticles formed by the self-assembly of berberine and isochlorogenic acid or structural analogs of berberine and isochlorogenic acid, and the specific steps include: S1: Dissolve berberine and isochlorogenic acid in a first solvent and adjust the pH to neutral to obtain a first mixed solution; the molar ratio of berberine to isochlorogenic acid is 1:1~4; then slowly add the first mixed solution dropwise to a second solvent, and set the temperature of the second solvent to 50℃-70℃; stir thoroughly to obtain a second mixed solution; The first solvent is an organic solvent, including one or more of dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, or tetrahydrofuran; the second solvent includes ultrapure water, phosphate buffer, or physiological saline. S2: Dialyze the second mixed solution to obtain the berberine and isochlorogenic acid nanomedicine.

8. The preparation method according to claim 7, characterized in that, The isochlorogenic acid includes isochlorogenic acid B, isochlorogenic acid A, or isochlorogenic acid C; the structural analogues of the isochlorogenic acid include 1,3,5-dicaffeoylquinic acid or 1,4,5-dicaffeoylquinic acid.

9. The preparation method according to claim 7, characterized in that, In S2, a dialysis bag is used to dialyze with ultrapure water for 12-48 hours. The molecular weight cutoff of the dialysis bag is 1000 Da-3000 Da.

10. The preparation method according to claim 7, characterized in that, In S1, a microfluidic device is used to introduce the first mixed solution and the second solvent into a Y-type mixer or a spiral mixing channel to mix them and obtain a second mixed solution; the flow rate ratio of the first mixed solution to the second solvent in the Y-type mixer or spiral mixing channel is 5-20:1, and the flow rate is 1-20 mL / h.

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