Nano-drug material based on self-assembly of berberine and caffeic acid and preparation method of nano-drug material

Abstract

The invention discloses a nano-drug material based on self-assembly of berberine and caffeic acid and a preparation method of the nano-drug material. The preparation method comprises the following steps: respectively dissolving berberine and caffeic acid in an organic solvent to prepare a berberine-organic solvent solution and a caffeic acid-organic solvent solution; adjusting the pH value of the caffeic acid-organic solvent solution to 7.0-7.5; adding the caffeic acid-organic solvent solution while stirring the berberine-organic solvent solution, uniformly mixing, adding the mixed solution into deionized water with the preheating temperature of 30-70 DEG C, and stirring at a constant temperature, so that the berberine and the caffeic acid are self-assembled to obtain a mixed solution; dialyzing the mixed solution to obtain a berberine and caffeic acid self-assembled solution; and performing vacuum freeze drying to obtain a target product. The nano-drug material disclosed by the invention is small in particle size, better in solubility and dissolution rate in different pH environments than berberine hydrochloride and caffeic acid eutectic, good in stability in an aqueous solution and environment-friendly in method.

Description

Technical Field

[0001] This invention relates to a Coptis chinensis drug, and more particularly to a Coptis chinensis-derived nanomedicine material and its preparation method, which is constructed by a self-assembly process using berberine and caffeic acid extracted from the traditional Chinese medicine Coptis chinensis as raw materials. Background Technology

[0002] In recent years, nanomedicine materials have become a research hotspot in the pharmaceutical field due to their advantages such as small particle size, large specific surface area, high bioavailability, and strong targeting potential, and are widely used in areas such as small molecule drug delivery and protection of bioactive substances. Currently, most mainstream nanomedicine materials on the market rely on chemically synthesized raw materials, which have problems such as high preparation costs, insufficient biocompatibility, and potential toxic side effects. On the other hand, nanomedicine materials developed based on natural products have gradually become a research focus due to their safe sources and good biocompatibility. However, the technology of extracting multiple active ingredients from a single traditional Chinese medicine and synergistically constructing nanomaterials is still relatively scarce, making it difficult to fully utilize the natural advantage of the "multi-component synergistic effect" of traditional Chinese medicine.

[0003] Coptis chinensis, a traditional Chinese medicine, has been used for over two thousand years. It possesses properties such as clearing heat and drying dampness, purging fire and detoxifying, and is widely used in the treatment of gastrointestinal diseases and infectious diseases. Modern pharmacological research confirms that the core medicinal value of Coptis chinensis stems from its various alkaloids and phenolic acids, among which berberine and caffeic acid are the two main components with the highest content and most clearly defined activity.

[0004] Berberine, an isoquinoline alkaloid, accounts for 5%-8% of the total active ingredients in Coptis chinensis. Its most significant effects are lowering blood sugar and regulating blood lipids. It effectively controls blood sugar by improving insulin resistance, inhibiting hepatic glucose output, and regulating gut microbiota, while simultaneously lowering cholesterol and triglycerides, thus earning it the nickname "natural metformin." Furthermore, berberine possesses strong antibacterial and anti-inflammatory properties and has traditionally been used to treat intestinal infections. It also has positive benefits for cardiovascular protection and improving fatty liver, and is a hallmark component of Coptis chinensis's efficacy.

[0005] Caffeic acid, a phenolic acid compound, accounts for 1%-3% of the total active ingredients in Coptis chinensis. Its core functions are potent antioxidant and anti-inflammatory effects, capable of scavenging harmful free radicals in the body and reducing oxidative stress damage, thereby helping the body resist aging and chronic diseases. Simultaneously, studies show it possesses certain antibacterial and antiviral capabilities. Clinically, its derivatives (such as caffeic acid tablets) are often used as hemostatic agents. Furthermore, caffeic acid has potential benefits in promoting bile secretion and protecting the cardiovascular system, and can help improve the activity, stability, and bioavailability of berberine.

[0006] Currently, research on the active ingredients of Coptis chinensis is mostly focused on "single component extraction and application" (such as the development of berberine monomer drugs), and has not fully utilized the natural correlation between "berberine and caffeic acid originating from Coptis chinensis", nor has it explored the technical path for the two to synergistically construct nanomedicine materials, resulting in the underutilization of Coptis chinensis' medicinal resources. The current state of research on single components has the following defects: (1) Raw material utilization: Traditional extraction processes of active ingredients of Coptis chinensis are mostly for single components (such as extracting only berberine), and other main components such as caffeic acid are discarded as by-products, resulting in the waste of Chinese medicine resources and the inability to utilize the synergistic effect between different components; (2) Material preparation: Existing Chinese medicine-derived nanomedicine materials mostly adopt the "single component loading" mode (such as loading berberine onto an external nanocarrier), which requires the introduction of additional carrier materials, resulting in complicated preparation steps, poor compatibility between components, and increased potential safety risks; (3) Performance: Drug preparations made from single berberine have problems such as poor water solubility and insufficient stability, while the antioxidant and membrane stabilizing effects of caffeic acid have not been fully utilized, and the material performance cannot be improved through component synergy.

[0007] Existing technologies also include some studies on the combination of components of Coptis chinensis, such as Chinese invention patent CN110054624B, which discloses a cocrystal of berberine hydrochloride and caffeic acid, its preparation method, composition, and uses. This technology uses berberine hydrochloride as the active pharmaceutical ingredient and caffeic acid as the cocrystal precursor to form a cocrystal. However, the cocrystal of this technology has a micron-scale crystal structure and lacks nanoscale features, failing to leverage the advantages of dispersibility and specific surface area brought by the nanostructure. Moreover, the molar ratio of berberine hydrochloride to caffeic acid in this technology is fixed at 2:1, lacking flexibility for adjustment and making it difficult to adapt to different application needs. The preparation process of this technology depends on specific crystal formation conditions, and the crystals are prone to agglomeration during storage and formulation processing, requiring additional pulverization and increasing the production process. This technology only improves solubility through crystal structure and does not fully utilize the long-term stability advantage brought by the nanostructure formed by the synergistic self-assembly of the two components.

[0008] Among existing similar technologies, there are also nanomedicines that combine a single Chinese herbal ingredient, Coptis chinensis, with a synthetic nanocarrier. For example, Chinese invention patent CN102949375B discloses a berberine hydrochloride solid lipid nanopreparation and its preparation method. This method uses solid lipids as a carrier to load berberine, which belongs to the category of nanomedicines combining berberine and synthetic nanocarriers. The preparation steps involve complex processes such as thin film dispersion and homogenization. This technology loads berberine onto artificially synthesized liposome carriers, relying on external synthetic carriers, which poses potential risks to biocompatibility. Furthermore, the preparation process requires the introduction of chemical reagents such as emulsifiers and crosslinking agents. Moreover, the preparation steps are cumbersome, involving multiple complex processes such as emulsification, ultrasound, and centrifugation, making large-scale production difficult. This technology involves the combination of active ingredients and carriers, and the interaction between the carrier and the active ingredient drug is only physical loading, which is prone to drug leakage and lacks stability. It also does not utilize the synergistic effect of naturally coexisting components in Chinese herbal medicines, resulting in low resource utilization. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies involving single Chinese herbal components of Coptis chinensis, synthetic nanocarrier drugs, and berberine hydrochloride and caffeic acid cocrystals, this invention provides a berberine and caffeic acid self-assembled nanomedicine and its preparation method, which features small particle size, superior solubility and dissolution rate under different pH conditions compared to berberine hydrochloride and caffeic acid cocrystals, good stability in aqueous solution, environmentally friendly preparation, and improved clinical applicability.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A method for preparing a self-assembled nanomedicine of berberine and caffeic acid includes the following steps:

[0012] (1) Berberine and caffeic acid are dissolved in organic solvents respectively to prepare berberine-organic solvent solution and caffeic acid-organic solvent solution; the molar ratio of berberine to caffeic acid is 1:2~2:1;

[0013] (2) Adjust the pH of the caffeic acid-organic solvent solution to 7.0~7.5;

[0014] (3) Add caffeic acid-organic solvent solution to berberine-organic solvent solution under stirring, mix well to obtain berberine and caffeic acid-organic solvent mixed solution;

[0015] (4) Add the mixed solution to deionized water preheated to 30~70℃, stir at a constant temperature to allow berberine and caffeic acid to self-assemble and obtain the mixed solution;

[0016] (5) Place the mixed solution in a dialysis bag and dialyze with ultrapure water for 36-60 hours to obtain a self-assembled solution of berberine and caffeic acid; the molecular weight cutoff of the dialysis bag is 3.0-4.0 kDa;

[0017] (6) Collect the self-assembled solution of berberine and caffeic acid, freeze-dry it under vacuum to obtain the self-assembled nanomedicine of berberine and caffeic acid.

[0018] To further achieve the purpose of this invention, preferably, in step (1), the concentration of berberine in the organic solvent is 1~5 mg / mL, and the concentration of caffeic acid in the organic solvent is 0.5~3 mg / mL; the organic solvent is selected from one or more of methanol, ethanol, and propanol.

[0019] Preferably, in step (2), the pH value of the caffeic acid-organic solvent solution is adjusted to 7.0~7.5 by adding an alkaline regulator.

[0020] Preferably, the alkalinity regulator is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, and sodium carbonate solution; the concentration of the alkalinity regulator is 5~20 mg / mL.

[0021] Preferably, in step (3), the stirring is achieved by a magnetic stirrer; the stirring speed is 600~1000 rpm.

[0022] Preferably, in step (3), the addition rate of the caffeic acid-organic solvent is 0.5~2 mL / min.

[0023] Preferably, in step (4), the constant temperature stirring time is 20 to 60 minutes; the volume ratio of the mixed solution to deionized water is 1:3 to 1:8.

[0024] Preferably, the ultrapure water is replaced every 6 to 10 hours during dialysis; the freeze-drying temperature is -85 to -75°C, the time is 10 to 24 hours, and the water is then freeze-dried in a freeze dryer for 40 to 60 hours after freezing.

[0025] A self-assembled nanomedicine of berberine and caffeic acid was prepared by the above-described method.

[0026] Preferably, the berberine and caffeic acid self-assembled nanomedicine has an average hydrated particle size of 100~1100nm, a polydispersity index (PDI) ≤0.75, a zeta potential of -35~-25mV, and a particle size fluctuation of ≤10% over 7 consecutive days in aqueous solution.

[0027] Compared with the prior art, the present invention has the following advantages and superior effects:

[0028] 1) The self-assembled nanomedicine of berberine and caffeic acid in this invention exhibits superior solubility and dissolution rate compared to the cocrystal of berberine hydrochloride and caffeic acid under different pH conditions. It can rapidly and stably release the drug components and is more suitable for the complex physiological environment in vivo. Although the cocrystal drug improves solid solubility, it lacks the support of nanostructures after dissolution, resulting in limited dissolution rate and dispersion uniformity. In contrast, the nanomedicine of this invention is itself a nanoscale dispersion. After entering the body, it does not require an additional "dissolution-dispersion" process and can directly contact target cells in the form of nanoparticles, resulting in higher efficiency.

[0029] 2) The self-assembled nanomedicine of berberine and caffeic acid of this invention can be stably maintained in the range of 140~150nm after being placed in an aqueous solution for 7 days. The polydispersity index (PDI) is always ≤0.3 and there is no obvious agglomeration phenomenon, which shows good stability. This stability advantage enables the nanomedicine to effectively avoid the loss of targeting and potential safety risks caused by the re-agglomeration of molecules after dissolution of cocrystal drugs to form large particles after dissolution. It solves the problems of insufficient stability and easy agglomeration of traditional berberine preparations.

[0030] 3) The particle size distribution of the self-assembled nanomedicine of berberine and caffeic acid in this invention is unimodal and symmetrical, with an average hydrated particle size of 100-1100 nm and a PDI of 0.2-0.3. The nanoparticles are uniform in size and dispersed evenly. The Zeta potential is -30.28 mV. The strong negative potential can generate electrostatic repulsion between nanoparticles, avoid particle aggregation, and thus ensure its stability in aqueous solution.

[0031] 4) This invention utilizes berberine and caffeic acid to self-assemble nanomedicines. The self-assembly process does not require additional chemical cross-linking agents, but is formed solely through the interaction between components, reducing foreign body irritation and toxic side effects on normal tissues. It also expands the routes of administration (such as intravenous injection and local administration) and improves clinical applicability.

[0032] 5) The berberine and caffeic acid self-assembled nanomedicine of this invention does not require the introduction of additional carrier materials, emulsifiers or cross-linking agents. It achieves self-assembly only through the natural interaction between the components. The steps are simple and easy to operate. Moreover, the organic solvent can be efficiently removed by dialysis, which is environmentally friendly and has the advantage of large-scale production.

[0033] 6) The raw materials of this invention are all derived from the natural Chinese herbal medicine Coptis chinensis, and there is no risk of chemical reagent residue in the preparation process. The biocompatibility is better than that of synthetic carrier-based nanomedicines. It simultaneously utilizes the two main active ingredients in Coptis chinensis, berberine and caffeic acid, to avoid the waste of resources caused by single-component extraction and give full play to the natural advantage of "multi-component synergy" of Chinese medicine. Attached Figure Description

[0034] Figure 1This is the assembly model of the self-assembled nanomedicine of berberine and caffeic acid of this invention.

[0035] Figure 2 This is an image of the aqueous solution of berberine, caffeic acid, and BBR-CAF NPs in Example 1.

[0036] Figure 3 This is a dynamic light scattering diagram of berberine and caffeic acid from Example 1, where A is the particle size diagram of BBR-CAF NPs and B is the zeta potential diagram of BBR-CAF NPs.

[0037] Figure 4 This is a graph showing the Yndall effect test results of the BBR-CAF NPs aqueous solution in Example 1.

[0038] Figure 5 This is an SEM image of the self-assembled nanoparticles (BBR-CAF NPs) of berberine and caffeic acid from Example 1.

[0039] Figure 6 This is a UV characterization result of the self-assembled nanoparticles of berberine and caffeic acid (BBR-CAF NPs) from Example 1.

[0040] Figure 7 This is an infrared characterization result of the self-assembled nanoparticles of berberine and caffeic acid (BBR-CAF NPs) in Example 1.

[0041] Figure 8 This is a test of the long-term stability of the berberine and caffeic acid self-assembled nanoparticles (BBR-CAF NPs) in Example 1. Detailed Implementation

[0042] To better understand the present invention, it will be further described below with reference to the accompanying drawings and specific embodiments. However, the implementation of the present invention is not limited thereto. The described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The key feature of this invention is the use of two key natural small-molecule active ingredients extracted from the single Chinese herb Coptis chinensis as core raw materials, and the preparation of nanomedicines through a specially designed self-assembly process. In existing technologies, berberine and caffeic acid are difficult to directly synergistically form nanomedicines, primarily due to their molecular properties and interaction characteristics. The structural formula of berberine is as follows: ;

[0044] Berberine is soluble in hot water, slightly soluble in water or ethanol, very slightly soluble in chloroform, and insoluble in ether.

[0045] The structural formula of caffeic acid is as follows: ;

[0046] Caffeic acid is an organic acid with the chemical formula C9H8O4. It is a yellow crystalline powder that is soluble in hot water and ethanol, and slightly soluble in cold water.

[0047] Generally speaking, berberine is an isoquinoline alkaloid with poor water solubility, with a solubility of only about 0.03 g / 100 mL in water. Although caffeic acid is a phenolic acid compound, its intermolecular forces are weak. Under normal conditions, the two can only mix simply through weak interactions and cannot spontaneously form stable nanoscale assemblies. If they are mixed directly, berberine is prone to precipitation and caffeic acid is prone to aggregation, making it impossible to maintain a nanoscale dispersion.

[0048] Therefore, existing technologies need to address the nanoscale problem of berberine, or the coordination problem between berberine and caffeic acid, to some extent through "carrier loading" or "eutectic".

[0049] For example, Chinese invention patent CN101683322B uses carrier technology to disperse berberine (or caffeic acid) into nano-sized particles by encapsulating / loading exogenous synthetic carriers (liposomes), thus solving the problems of poor water solubility and inability to disperse stably. However, it requires the introduction of synthetic carriers and chemical reagents, which poses a risk of biocompatibility.

[0050] For example, Chinese invention patent CN110054624B improves the solubility of berberine by forming a eutectic crystal through strong hydrogen bonding between berberine and caffeic acid molecules. The dissolution rate of the eutectic in water is significantly higher than that of the berberine raw material. However, the formation of the eutectic depends on the "crystallization" process of strong intermolecular interactions and cannot form a nanoscale structure. This is because the preparation of the eutectic in this technology uses conventional eutectic preparation processes such as "solvent evaporation" and "grinding". The products of such processes are usually micron-sized crystal particles. It is generally believed in the field that the particle size of eutectic prepared by solvent evaporation and grinding is mostly concentrated in the range of 1~100μm. In fact, this technology does not use nanoscale characterization methods such as dynamic light scattering and electron microscopy, but only uses "powder X-ray diffraction (XRD)" to characterize the eutectic structure. This method targets the crystal form characteristics of the crystal, rather than the nanoscale particle size.

[0051] This invention uses berberine and caffeic acid extracted simultaneously from Coptis chinensis as raw materials to synergistically self-assemble nanomedicine materials, effectively solving the problem that existing technologies cannot achieve the combination of berberine and caffeic acid and the preparation of nanomedicines. Figure 1This is an assembly model diagram of the self-assembled nanomedicine of berberine and caffeic acid according to the present invention. The aromatic ring system in the berberine molecule can form π-π stacking interactions with the benzene ring in the caffeic acid molecule. Simultaneously, stable hydrogen bonds can form between the carboxyl and phenolic hydroxyl groups in the caffeic acid molecule and the methoxy oxygen atom in the berberine molecule. These two non-covalent interactions work synergistically to drive the orderly aggregation of berberine and caffeic acid molecules, self-assembling into a stable nanoparticle structure. This assembly process requires no external force or chemical reagent assistance, relying solely on the natural interactions between the components, ensuring the biocompatibility and structural stability of the nanomedicine. Specifically, the preparation method of the self-assembled nanomedicine of berberine and caffeic acid according to the present invention comprises the following steps:

[0052] (1) Berberine and caffeic acid are dissolved in organic solvents respectively to prepare berberine-organic solvent solution and caffeic acid-organic solvent solution; the molar ratio of berberine to caffeic acid is 1:2~2:1;

[0053] (2) Adjust the pH of the caffeic acid-organic solvent solution to 7.0~7.5;

[0054] (3) Add caffeic acid-organic solvent solution to berberine-organic solvent solution under stirring, mix well to obtain berberine and caffeic acid-organic solvent mixed solution;

[0055] (4) Add the mixed solution to deionized water preheated to 30~70℃, stir at a constant temperature to allow berberine and caffeic acid to self-assemble and obtain the mixed solution;

[0056] (5) Place the mixed solution in a dialysis bag and dialyze with ultrapure water for 36-60 hours to obtain a self-assembled solution of berberine and caffeic acid; the molecular weight cutoff of the dialysis bag is 3.0-4.0 kDa;

[0057] (6) Collect the self-assembled solution of berberine and caffeic acid, freeze-dry it under vacuum to obtain the self-assembled nanomedicine of berberine and caffeic acid.

[0058] Regarding the selection and concentration of organic solvents for berberine and caffeic acid, based on the different solubilities of these two components, the preferred concentration of berberine in organic solvents is 1~5 mg / mL, and the preferred concentration of caffeic acid in organic solvents is 0.5~3 mg / mL; all organic solvents are preferably one or more of methanol, ethanol, and propanol.

[0059] Due to the acidic nature of caffeic acid, the pH of the caffeic acid-organic solvent solution is adjusted to 7.0-7.5 by adding an alkaline adjuster. Generally, one or more of sodium hydroxide solution, potassium hydroxide solution, and sodium carbonate solution are commonly used as alkaline adjusters; the concentration of the alkaline adjuster is generally 5-20 mg / mL.

[0060] For ease of operation, the stirring method of this invention is magnetic stirring; the stirring speed is preferably 600~1000 rpm.

[0061] Through testing, the preferred addition rate of caffeic acid-organic solvent is 0.5~2 mL / min.

[0062] The preferred stirring time at constant temperature is 20 to 60 minutes; the preferred volume ratio of the mixed solution to deionized water is 1:3 to 1:8.

[0063] After testing, it is recommended that the ultrapure water be replaced every 6 to 10 hours during dialysis in this invention; the freeze-drying temperature is preferably -85 to -75°C, the time is 10 to 24 hours, and after freeze-drying, it is freeze-dried again in a freeze dryer for 40 to 60 hours.

[0064] In the prior art, the berberine and caffeic acid cocrystal drug in Chinese invention patent CN110054624B relies on strong intermolecular hydrogen bonds to form a fixed crystal structure, and the crystal surface energy is high. During storage and processing, it is prone to agglomeration due to van der Waals forces, resulting in uncontrollable morphology and size. The present invention relies on the dynamic equilibrium of weak interactions (hydrogen bonds, π-π stacking), and the particle size (100-1100nm) and morphology (spherical) of the nanoparticles can be controlled by process parameters (such as molar ratio and temperature). Moreover, the electrostatic repulsion at the nanoscale (Zeta potential -35~-25mV) can effectively inhibit agglomeration and achieve long-term stability.

[0065] The caffeic acid (CAS No.: 331-39-5) and berberine hydrochloride (CAS No.: 633-65-8) used in the examples were all commercially available raw materials. The dialysis bags, organic solvents, and alkalinity adjusters used were all commonly used reagents in testing laboratories and could be obtained through commercial channels. The raw materials used in the examples of this invention are commercially available raw materials.

[0066] The particle size, PDI, and Zeta potential testing methods involved in the examples are as follows: referring to the national standard GB / T 30100-2013 "Dynamic Light Scattering Method for Nanoparticle Size Measurement", a Malvern particle size analyzer (model: Zetasizer ULTRA) was used for detection. The test temperature was 25℃, and each sample was tested in parallel 3 times, and the average value was taken. Before the test, the sample was diluted with ultrapure water to a suitable concentration to ensure the stability of the test signal.

[0067] It should be noted that, according to the National Medical Products Administration's "Technical Guidelines for Quality Control Research of Nanomedicines (Trial)" and international consensus in the pharmaceutical field, the scope of nanomedicines / nanodelivery systems typically covers particle systems in the 1-1000 nm range. Drug carriers / formulations within this scale range can exhibit the core effects unique to the nanoscale (such as enhanced permeation retention (EPR), improved dissolution rate, and improved dispersion stability), and are recognized as the category of "nanomedicines" in practical applications in the pharmaceutical field.

[0068] Example 1: Preparation of self-assembled nanomedicine from berberine and caffeic acid (molar ratio 1:1, moderate process parameters)

[0069] A method for preparing a self-assembled nanomedicine of berberine and caffeic acid includes the following steps:

[0070] (1) Weigh 38 mg of berberine and 18 mg of caffeic acid in a molar ratio of 1:1, and dissolve them in 10 mL of methanol to prepare berberine-methanol solution (concentration 3.8 mg / mL) and caffeic acid-methanol solution (concentration 1.8 mg / mL).

[0071] (2) Adjust the pH of the caffeic acid-methanol solution to 7.2 using a 5 mg / mL sodium hydroxide solution;

[0072] (3) Place the berberine-methanol solution under a magnetic stirrer and stir at 800 rpm. At the same time, add the caffeic acid-methanol solution dropwise to the berberine-methanol solution at a rate of 1 mL / min. After the addition is complete, continue stirring for 8 minutes to mix evenly.

[0073] (4) The above mixed solution was slowly added dropwise to 10 mL of deionized water preheated to 60 °C, and stirred at a constant temperature for 30 minutes to allow berberine and caffeic acid to self-assemble and obtain a mixed solution.

[0074] (5) The mixed solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with 500 mL of ultrapure water for 48 h. The ultrapure water was changed every 8 h to obtain a berberine-caffeic acid self-assembled solution.

[0075] (6) The self-assembled solution was collected, frozen at -80℃ for 12h, and then freeze-dried in a freeze dryer for 48h to obtain yellow nanoparticle freeze-dried powder.

[0076] Performance characterization and results analysis:

[0077] Appearance comparison analysis (Figure 2)

[0078] Figure 2 shows a comparison of the appearance of berberine aqueous solution (A), caffeic acid aqueous solution (B), and berberine and caffeic acid self-assembled nanomedicine (BBR-CAF NPs) aqueous solution (C) in Example 1. Preparation method: Berberine, caffeic acid, and the berberine and caffeic acid self-assembled nanomedicine prepared in this example were each prepared into a 0.5 mg / mL solution using ultrapure water (all three had the same concentration). Images were taken after standing at room temperature for 1 hour. Figure 2 The results showed that the berberine aqueous solution (A) was a pale yellow turbid liquid with a small amount of precipitate after standing; the caffeic acid aqueous solution (B) was a light brown translucent liquid with slight turbidity; while the BBR-CAF NPs aqueous solution (C) was a uniform golden yellow transparent liquid with no precipitation or layering. These results indicate that the self-assembly of nanostructures significantly improves the dispersibility of berberine in water, solving its poor water solubility and easy precipitation problems, and providing a foundation for its leaching applications.

[0079] Dynamic light scattering analysis (Figure 3)

[0080] Figure 3 shows the dynamic light scattering test results of the BBR-CAF NPs prepared in Example 1. In the figure, A is the particle size distribution spectrum, and B is the Zeta potential spectrum. The test method followed GB / T 30100-2013 "Dynamic Light Scattering Method for Nanoparticle Size Measurement". The lyophilized powder of the self-assembled nanomedicine of berberine and caffeic acid was diluted 20 times with ultrapure water, placed in a particle size analyzer, and measured at 25℃ using a Malvern particle size analyzer. Figure A shows that the particle size distribution of the self-assembled nanomedicine of berberine and caffeic acid exhibits a unimodal symmetrical distribution, with an average hydrated particle size of 142.1 nm and a PDI of 0.2438, indicating that the nanoparticles are uniform in size and dispersed evenly. Figure B shows that the Zeta potential is -30.28 mV. The strong negative potential can generate electrostatic repulsion between nanoparticles, preventing particle aggregation and ensuring its stability in aqueous solution.

[0081] In existing technologies, berberine and caffeic acid cocrystal drugs (CN110054624B), as micron-sized powders, tend to re-aggregate into large particles after dissolving in the body (e.g., blood, intestinal fluid) due to a lack of nanoscale electrostatic repulsion / steric hindrance. This results in poor solution stability, affecting the long-term storage and efficacy of the formulation. Furthermore, the micron-sized powder or crystals of the cocrystal drug (CN110054624B) limit the route of administration (mostly oral), making it difficult to meet the clinical demand for highly efficient delivery methods such as injections. In contrast, the nanoscale (especially 100-200 nm) particles of this invention can utilize the unique "enhanced permeability and long-term retention effect (EPR effect)" of tumor tissue to passively target and accumulate at the tumor site. This significantly increases the concentration of the nanomedicine at the lesion. The nanoscale size avoids vascular embolism, and the self-assembly process does not require additional chemical cross-linking agents, relying solely on the interaction between components. This reduces foreign body irritation and lowers toxic side effects on normal tissues, expanding the route of administration (e.g., intravenous injection, local administration) and improving clinical applicability.

[0082] Tyndall effect analysis (Figure 4)

[0083] Figure 4 shows the Tyndall effect test results of the BBR-CAF NPs aqueous solution. Test method: The BBR-CAF NPs aqueous solution prepared in Example 1 was placed in a transparent test tube and illuminated from the side of the test tube using a 5mW laser pointer in a dark room. The scattered light phenomenon was observed. As shown in the figure, a clear bright "pathway" was formed when the laser beam passed through the solution, indicating a significant Tyndall effect. This phenomenon confirms the existence of stably dispersed nanoscale particles in the aqueous solution, further demonstrating that berberine and caffeic acid successfully self-assembled into a nanostructure, consistent with the dynamic light scattering test results. During the self-assembly process, the ratio of berberine to caffeic acid can be flexibly adjusted (e.g., 1:1, 2:1, 1:2), and the synergistic effects of "antibacterial + anti-inflammatory" and "hypoglycemic + lipid-lowering" can be enhanced by optimizing the ratio. The nanoparticles can simultaneously load two components, achieving "synchronous delivery and synergistic effect." This invention addresses the problem of existing berberine hydrochloride and caffeic acid cocrystal drugs (CN110054624B) having a fixed molar ratio (2:1), which cannot meet the synergistic needs of different diseases, thereby improving the targeted nature of treatment.

[0084] Scanning electron microscopy (SEM) analysis (Figure 5)

[0085] Figure 5 shows the SEM image of the BBR-CAF NPs prepared in Example 1. Sample preparation: The dialyzed BBR-CAF NPs solution was diluted 50 times, and 10 μL was dropped onto a silicon wafer. The wafer was then dried naturally at room temperature, fixed onto conductive adhesive, and sputter-coated with gold. The sample was then observed using a SU8600 scanning electron microscope at an accelerating voltage of 5.00 kV. As shown in the figure, the nanoparticles exhibit a uniform spherical morphology with no obvious agglomeration. The particle size is consistent with the dynamic light scattering test results, indicating that the prepared nanomedicine possesses ideal nanoparticle morphology characteristics, providing structural assurance for its dispersion stability and application performance.

[0086] Ultraviolet characterization analysis (Figure 6)

[0087] Figure 6 shows the UV characterization results of BBR, CAF, and BBR-CAF NPs. Test method: BBR, CAF, and BBR-CAF NPs were dissolved in ultrapure water to prepare solutions with a concentration of 0.1 mg / mL. 3 mL of each solution was placed in a cuvette, and the samples were detected using a UV spectrophotometer in the wavelength range of 200–500 nm. Figure 6 It was observed that BBR exhibited characteristic absorption peaks at 425 nm and 345 nm, while CAF showed a characteristic absorption peak at 320 nm. The BBR-CAF NPs, however, exhibited characteristic absorption peaks at 330 nm, 275 nm, and 425 nm. These peak positions were related to, but slightly offset from, the characteristic absorption peaks of BBR and CAF, indicating that they were not simply superimposed. This result confirms that an interaction occurred between berberine and caffeic acid, forming a novel supramolecular nanocomposite, rather than a physical mixture.

[0088] External characterization analysis (Figure 7)

[0089] Figure 7 shows the infrared characterization results of BBR, CAF, and BBR-CAF NPs. Test method: 2 mg each of BBR, CAF, and freeze-dried BBR-CAF NPs were weighed and added to 200 mg of infrared-dried potassium bromide powder. The mixture was ground and pressed into potassium bromide tablets, and detected using an infrared spectrometer in the range of 4000–400 cm⁻¹. The spectrometer resolution was 4 cm⁻¹, the signal-to-noise ratio was 50000:1, and 64 scans were performed. Figure 7It can be seen that in the BBR-CAF NP spectrum, the OH stretching vibration peak (~3300 cm⁻¹) is broader and weaker than the corresponding peak of CAF, while the C=O stretching vibration peak (~1700 cm⁻¹) shifts to a lower wavenumber by about 10 cm⁻¹. This phenomenon indicates that a strong hydrogen bond is formed between the carboxyl / phenolic hydroxyl group of caffeic acid and the methoxy oxygen atom or aromatic ring system of berberine, leading to a change in the chemical bond force constant. This further confirms that berberine and caffeic acid successfully self-assemble into a nanocomposite through non-covalent interactions.

[0090] Long-term stability analysis (Figure 8)

[0091] Figure 8 shows the long-term stability test results of BBR-CAF NPs. Test method: The BBR-CAF NPs prepared in Example 1 were dispersed in ultrapure water to prepare a solution with a concentration of 1 mg / mL. The solution was sealed and placed at room temperature. Particle size and PDI were measured using a Malvern particle size analyzer on days 1, 3, 5, and 7. As shown in the figure, during the 7-day placement period, the average particle size of the nanomedicine remained stably within the range of 140–150 nm, and the PDI remained between 0.2 and 0.3, without significant fluctuations.

[0092] The berberine and caffeic acid cocrystal drug (CN110054624B) was tested for its chemical stability in the solid state (by XRD to detect changes in crystal structure) under the scenario of "storage stability under high temperature / high humidity / light / pressure," addressing the issue of "non-degradation and non-crystallization during solid drug storage." Compared to cocrystal drugs, which focus on chemical stability during solid storage, the nanomedicine of this invention demonstrates superior colloidal stability in an aqueous solution simulating the body's fluid environment through dynamic light scattering (DLS) testing. Specifically, at room temperature, after being continuously placed in an aqueous solution for 7 days, the nanomedicine's particle size remained stably within the range of 140-150 nm, with a polydispersity index (PDI) consistently ≤0.3, and no significant aggregation was observed. This stability advantage effectively avoids the targeting loss and potential safety risks caused by the re-aggregation of molecules into large particles after dissolution in cocrystal drugs. Meanwhile, the nanomedicines do not require an additional dissolution-dispersion process and can directly and rapidly contact target cells in nanoparticle form, significantly improving the drug's efficacy. These results indicate that the berberine-caffeic acid self-assembled nanomedicines prepared in this invention possess good long-term stability, can exist stably in aqueous solution, and meet the basic requirements for subsequent storage and application.

[0093] Comparative test: Dissolution rate comparison with eutectic drug (CN110054624B)

[0094] Test method: Referring to the dissolution test method (paddle method, 50 rpm, 37 ℃) in the Chinese Pharmacopoeia, 50 mg of each of the nano-drug (BBR-CAF NPs) and cocrystal drug (YSXBJ-KFS, data source: comparative file CN110054624B) prepared in Example 1 of this invention were placed in 900 mL of buffer solution with corresponding pH values, and the dissolution rate of berberine at different time points was measured. The test results are shown in Table 1.

[0095] Table 1 ;

[0096] In pH=1.0 (simulating gastric acid environment): the cocrystallized drug had a dissolution rate of only 38.4% after 30 minutes, and the dissolution rate dropped after 60 minutes; while the nano-drug of this invention had a dissolution rate of 90.5% after 30 minutes and was almost completely dissolved after 120 minutes, which solved the problem of slow and unstable dissolution of cocrystallized drugs in acidic environments.

[0097] At pH=6.8 (simulating the intestinal environment): the dissolution rate of the cocrystallized drug was 93.5% after 30 minutes, and the dissolution rate of the nano-drug of this invention reached 99.1% after 30 minutes. It can achieve the dissolution level of the cocrystallized drug after 30 minutes within 10 minutes, which significantly improves the dissolution rate and completeness.

[0098] Table 1 shows that the self-assembled nanomedicine of berberine and caffeic acid exhibits superior solubility and dissolution behavior compared to the cocrystal drug under different pH conditions. It can rapidly and stably release the drug components, making it more suitable for the complex physiological environment in vivo. While the cocrystal drug improves solid solubility, it lacks the support of a nanostructure after dissolution, resulting in limited dissolution rate and dispersion uniformity. In contrast, the nanomedicine of this invention is itself a nanoscale dispersion, eliminating the need for an additional "dissolution-dispersion" process after entering the body. It can directly contact target cells in the form of nanoparticles, leading to higher efficacy.

[0099] The characterization test figures in the following embodiments are basically the same as those in Embodiment 1, and are not provided one by one; only the relevant test data are given.

[0100] Example 2: Preparation of self-assembled nanomedicines of berberine and caffeic acid (molar ratio 1:2, low concentration solvent parameters)

[0101] A method for preparing a self-assembled nanomedicine of berberine and caffeic acid includes the following steps:

[0102] (1) Weigh 19 mg of berberine and 18 mg of caffeic acid in a molar ratio of 1:2, and dissolve them in 10 mL of ethanol to prepare berberine-ethanol solution (concentration 1.9 mg / mL) and caffeic acid-ethanol solution (concentration 1.8 mg / mL).

[0103] (2) Adjust the pH of the caffeic acid-ethanol solution to 7.0 using a 3 mg / mL potassium hydroxide solution;

[0104] (3) Place the berberine-ethanol solution under a magnetic stirrer and stir at a speed of 600 rpm. At the same time, add the caffeic acid-ethanol solution dropwise to the berberine-ethanol solution at a rate of 0.5 mL / min. After the addition is complete, continue stirring for 5 minutes to mix evenly.

[0105] (4) Slowly add the above mixed solution dropwise to 8 mL of deionized water preheated to 40 °C, and stir at a constant temperature for 20 minutes to allow berberine and caffeic acid to self-assemble and obtain a mixed solution;

[0106] (5) Place the mixed solution in a dialysis bag with a molecular weight cutoff of 3.0 kDa, and dialyze with 500 mL of ultrapure water for 36 h, changing the ultrapure water every 6 h to obtain a self-assembled solution of berberine and caffeic acid.

[0107] (6) The self-assembled solution was collected, frozen at -75℃ for 10h, and then freeze-dried in a freeze dryer for 40h to obtain yellow nanoparticle freeze-dried powder.

[0108] The nanomedicine was tested and found to have an average hydrated particle size of 617.8 nm, a PDI of 0.4879, a Zeta potential of -28.5 mV, and a particle size that remained within the range of 580-650 nm for 7 consecutive days in aqueous solution.

[0109] Example 3: Preparation of self-assembled nanomedicine from berberine and caffeic acid (molar ratio 2:1, high-temperature process parameters)

[0110] A method for preparing a self-assembled nanomedicine of berberine and caffeic acid includes the following steps:

[0111] (1) Weigh 76 mg of berberine and 18 mg of caffeic acid in a molar ratio of 2:1, and dissolve them in 15 mL of propanol to prepare berberine-propanol solution (concentration 5.1 mg / mL) and caffeic acid-propanol solution (concentration 1.2 mg / mL).

[0112] (2) Adjust the pH of the caffeic acid-propanol solution to 7.5 using an 8 mg / mL sodium carbonate solution;

[0113] (3) Place the berberine-propanol solution under a magnetic stirrer and stir at 1000 rpm. At the same time, add the caffeic acid-propanol solution dropwise to the berberine-propanol solution at a rate of 2 mL / min. After the addition is complete, continue stirring for 10 minutes to mix evenly.

[0114] (4) The above mixed solution was slowly added dropwise to 15 mL of deionized water preheated to 80 °C, and stirred at a constant temperature for 40 minutes to allow berberine and caffeic acid to self-assemble and obtain a mixed solution.

[0115] (5) Place the mixed solution in a dialysis bag with a molecular weight cutoff of 4.0 kDa, dialyze with 500 mL of ultrapure water for 60 h, and change the ultrapure water every 10 h to obtain a berberine-caffeic acid self-assembled solution.

[0116] (6) The self-assembled solution was collected, frozen at -85℃ for 14h, and then freeze-dried in a freeze dryer for 50h to obtain yellow nanoparticle freeze-dried powder.

[0117] The nanomedicine was tested and found to have an average hydrated particle size of 1016 nm, a PDI of 0.7164, a Zeta potential of -32.8 mV, and a particle size that remained within the range of 950-1080 nm for 7 consecutive days in aqueous solution.

[0118] As can be seen from the preparation method of this invention, this invention uses berberine and caffeic acid, two main active ingredients extracted from Coptis chinensis, as the main raw materials. Through the synergistic reaction of the two, nanomedicine materials are constructed, which not only retain the natural activity of the traditional Chinese medicine ingredients, but also give full play to the advantages of the nanostructure, improve the solubility and bioavailability of the drug, and further enhance its drug effect.

[0119] Moreover, this invention does not require the introduction of additional carrier materials, which can not only achieve efficient utilization of traditional Chinese medicine resources, but also solve the defects of existing nanomedicine materials such as complex preparation and insufficient stability. The self-assembled nanomedicine provided by this invention has a flexible adjustment of the molar ratio of the components, which enhances synergistic biological activity; good dispersion stability, simple formulation processing; adaptable to multiple administration routes, and high safety.

[0120] As can be seen from Examples 1-3, in the preparation method of the self-assembled nanomedicine of berberine and caffeic acid of the present invention, the molar ratio of berberine to caffeic acid is 1:2 to 2:1, and the average hydrated particle size and PDI value of the obtained nanomedicine are shown in Table 2.

[0121] Table 2. Average particle size and PDI values ​​of nanomedicine synthesized with different molar ratios of berberine and caffeic acid. ;

[0122] As shown in Table 2, the molar ratio of berberine to caffeic acid in the raw materials can be flexibly controlled within the range of 1:2 to 2:1, and both can self-assemble to form stable nanomedicines. When the molar ratio is 1:1, the prepared nanomedicine has the smallest average hydrated particle size (142.1 nm) and a PDI as low as 0.2438 (≤0.3, which meets the evaluation criteria for uniform distribution of nanomedicines). It has a narrow particle size distribution and excellent stability, and can exist stably in bodily fluid environments such as blood. When the molar ratio is 1:2 (particle size 617.8 nm, PDI 0.4879) or 2:1 (particle size 1016 nm, PDI 0.7164), although the particle size and dispersibility are slightly inferior to the 1:1 ratio, they are still within the range of nanomedicines recognized in the pharmaceutical field (1~1000 nm), and can maintain basic dispersion stability. The molar ratio of berberine hydrochloride-caffeic acid cocrystal disclosed in Chinese invention patent CN110054624B is strictly fixed at 2:1, making it impossible to adjust the component ratio according to the drug administration scenario (such as the synergistic activity requirements of different diseases). This invention breaks through this limitation through a self-assembly process, achieving flexible adaptation of molar ratios from 1:2 to 2:1, while preserving the synergistic effect between components and ensuring the basic stability of the nanomedicine, thus solving the technical limitation of a single molar ratio in cocrystal drugs.

[0123] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a self-assembled nanomedicine of berberine and caffeic acid, characterized in that... Includes the following steps: (1) Berberine and caffeic acid are dissolved in organic solvents respectively to prepare berberine-organic solvent solution and caffeic acid-organic solvent solution; the molar ratio of berberine to caffeic acid is 1:2~2:1; (2) Adjust the pH of the caffeic acid-organic solvent solution to 7.0~7.5; (3) Add caffeic acid-organic solvent solution to berberine-organic solvent solution under stirring, mix well to obtain berberine and caffeic acid-organic solvent mixed solution; (4) Add the mixed solution to deionized water preheated to 30~70℃, stir at a constant temperature to allow berberine and caffeic acid to self-assemble and obtain the mixed solution; (5) Place the mixed solution in a dialysis bag and dialyze with ultrapure water for 36-60 hours to obtain a self-assembled solution of berberine and caffeic acid; the molecular weight cutoff of the dialysis bag is 3.0-4.0 kDa; (6) Collect the self-assembled solution of berberine and caffeic acid, freeze-dry it under vacuum to obtain the self-assembled nanomedicine of berberine and caffeic acid.

2. The method for preparing berberine and caffeic acid self-assembled nanomedicine according to claim 1, characterized in that, In step (1), the concentration of berberine in the organic solvent is 1~5 mg / mL, and the concentration of caffeic acid in the organic solvent is 0.5~3 mg / mL; the organic solvent is selected from one or more of methanol, ethanol, and propanol.

3. The method for preparing berberine and caffeic acid self-assembled nanomedicine according to claim 1, characterized in that, In step (2), the pH value of the caffeic acid-organic solvent solution is adjusted to 7.0~7.5 by adding an alkaline regulator.

4. The method for preparing berberine and caffeic acid self-assembled nanomedicine according to claim 3, characterized in that, The alkalinity regulator is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, and sodium carbonate solution; the concentration of the alkalinity regulator is 5~20 mg / mL.

5. The method for preparing berberine and caffeic acid self-assembled nanomedicine according to claim 1, characterized in that, In step (3), the stirring is achieved by a magnetic stirrer; the stirring speed is 600~1000 rpm.

6. The method for preparing berberine and caffeic acid self-assembled nanomedicine according to claim 1, characterized in that, In step (3), the addition rate of the caffeic acid-organic solvent is 0.5~2 mL / min.

7. The method for preparing berberine and caffeic acid self-assembled nanomedicine according to claim 1, characterized in that, In step (4), the constant temperature stirring time is 20 to 60 minutes; the volume ratio of the mixed solution to deionized water is 1:3 to 1:

8.

8. The method for preparing berberine and caffeic acid self-assembled nanomedicine according to claim 1, characterized in that, The ultrapure water is replaced every 6 to 10 hours during dialysis; the freeze-drying temperature is -85 to -75°C, the time is 10 to 24 hours, and after freezing, it is freeze-dried in a freeze dryer for 40 to 60 hours.

9. A self-assembled nanomedicine of berberine and caffeic acid, characterized in that, It is prepared by the preparation method described in any one of claims 1-8.

10. The berberine and caffeic acid self-assembled nanomedicine according to claim 9, characterized in that, The berberine and caffeic acid self-assembled nanomedicine has an average hydrated particle size of 100~1100nm, a polydispersity index (PDI) ≤0.75, a zeta potential of -35~-25mV, and a particle size fluctuation of ≤10% over 7 consecutive days in aqueous solution.

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