Berberine nanometer material and preparation method and application thereof

Abstract

The invention discloses a berberine nano material as well as a preparation method and application thereof, and belongs to the technical field of nano materials. According to the preparation method provided by the invention, EGCG and BBR are combined with a specific nano-carrier to obtain the berberine nano-material capable of remarkably improving the water solubility of berberine and greatly improving the bioavailability of the berberine, and then the berberine nano-material can be accurately enriched at a renal lesion part through a targeted modification technology, so that the bioavailability of the berberine is greatly improved. The treatment effect on kidney diseases is enhanced, and meanwhile, the distribution of medicines in other organs is reduced, so that the side effects are reduced. The preparation method disclosed by the invention is simple and convenient, industrial production is convenient to realize, and the prepared product provides an efficient, safe and economical novel drug delivery scheme for clinical treatment of kidney diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and in particular relates to a berberine nanomaterial and a preparation method and application thereof. Background Art

[0002] Kidney disease poses a serious threat to human health, with its incidence increasing year by year. Common kidney diseases, such as acute kidney injury and chronic kidney disease, not only cause significant suffering to patients but also place a heavy burden on the healthcare system. Currently, treatment options for kidney disease are limited, and traditional medications have numerous deficiencies in efficacy and safety. Berberine, an isoquinoline alkaloid extracted from various Chinese herbs such as Coptis chinensis and Phellodendron amurense, possesses a wide range of pharmacological activities, including antibacterial, anti-inflammatory, antioxidant, and blood sugar and lipid regulation. Berberine has shown promising potential in the treatment of kidney disease. Studies have shown that it can protect the kidneys through various pathways, such as inhibiting the release of inflammatory factors, alleviating oxidative stress damage, and regulating cell apoptosis. However, berberine faces serious limitations in its practical application. First, its poor water solubility makes it difficult to dissolve and absorb in the body, resulting in extremely low bioavailability. Studies have shown that after oral administration of berberine, its blood concentration is difficult to reach an effective therapeutic level, and most of the drug is excreted without being absorbed. Furthermore, berberine lacks targeted targeting to the kidneys. During systemic distribution, it not only produces unwanted side effects in other organs but also reduces the drug dose reaching the affected kidneys, preventing it from fully exerting its therapeutic effect. To address drug solubility and renal targeting issues, nanomaterial technology has been widely researched and applied in the field of drug delivery. Nanomaterials exhibit unique small size, surface, and quantum size effects, which can improve the physicochemical properties of drugs, enhancing their stability, solubility, and bioavailability. By encapsulating or attaching drugs to nanocarriers, targeted drug delivery can be achieved, increasing drug accumulation at lesions and minimizing toxic side effects in normal tissues. Currently, a variety of nano-drug delivery systems, such as liposomes, polymer nanoparticles, and nanomicelles, have been developed for the treatment of renal diseases. However, most of these nanomaterials suffer from complex preparation processes, high costs, poor biodegradability, and potential toxicity. For example, some polymer nanoparticles are difficult to degrade in the body, and long-term accumulation may pose potential health risks. The preparation of some nanocarriers requires the use of large amounts of organic solvents, which can result in drug residues and compromise drug safety. Summary of the Invention

[0003] In response to the above-mentioned existing technologies, the present invention provides a berberine nanomaterial, a preparation method and application thereof, which solves the problems in the existing technology that berberine is difficult to fully exert its therapeutic effect on kidney diseases due to its poor water solubility, low bioavailability and lack of kidney targeting; at the same time, nanomaterials used for drug delivery have problems such as complex preparation process, high cost and poor safety.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing berberine nanomaterials, comprising the following steps: S1: Disperse the ZIF material in water to obtain a ZIF suspension, and then dissolve EGCG and BBR in DMSO to obtain EGCG solution and BBR solution respectively; S2: Add EGCG solution and BBR solution to the ZIF suspension to obtain a mixed solution, then incubate the mixed solution at 35-40°C and 200-300 rpm for 6-8 hours, followed by dialysis for 24-36 hours. S3: The dialyzed solution is centrifuged, the precipitate is washed, and the precipitate is freeze-dried to obtain the berberine nanomaterial.

[0005] The beneficial effects of the present invention are: the present invention provides a preparation method of berberine nanomaterials, which combines berberine with specific nanocarriers through an innovative synthesis process, significantly improves the water solubility of berberine, greatly improves its bioavailability, and uses targeted modification technology of nanomaterials to enable berberine nanomaterials to be accurately enriched in the lesion site of the kidney, while enhancing the therapeutic effect on kidney disease, reducing the distribution of the drug in other organs, thereby reducing side effects; and the synthesis method of the present invention is simple to operate and cost-controllable, and the raw materials and preparation process used meet safety requirements, which facilitates industrial production, and provides an efficient, safe and economical new drug delivery solution for the clinical treatment of kidney disease.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows.

[0007] Furthermore, the ZIF material is ZIF-8, ZIF-67 or ZIF-68.

[0008] The beneficial effects of adopting further technical solutions are: ZIF-8 has good biocompatibility, high specific surface area and controllable pore structure, and can load a large number of drug molecules; ZIF-67 can respond to the ROS environment at the lesion and synergistically promote drug release; ZIF-68 can further optimize its pH response performance through structural modification on the basis of maintaining framework stability; these ZIF materials can all degrade in a weakly acidic environment to achieve pH-responsive drug release.

[0009] Furthermore, freeze drying is performed at -50°C and a vacuum degree lower than 10 Pa for 25 to 30 hours.

[0010] The present invention also provides a berberine nano material prepared by the preparation method of the berberine nano material.

[0011] Furthermore, the mass fraction of EGCG in berberine nanomaterials is 10~20%.

[0012] Furthermore, the mass fraction of BBR in the berberine nanomaterial is 30~40%.

[0013] The beneficial effects of adopting further technical solutions are: as an auxiliary therapeutic ingredient, EGCG has strong antioxidant and anti-inflammatory capabilities, can effectively remove reactive oxygen species, inhibit the expression of inflammatory factors, reduce kidney tissue damage, and synergize with BBR to exert a therapeutic effect; as the main therapeutic ingredient, BBR has multiple biological activities such as regulating lipid metabolism, anti-inflammatory, and antioxidant.

[0014] The present invention also provides the use of berberine nanomaterials in the preparation of kidney-targeted drugs.

[0015] The beneficial effects of the present invention are as follows: the berberine nanomaterial provided by the present invention uses ZIF material as a core carrier to provide space for drug loading, BBR is the main active ingredient for treating diabetic nephropathy, and EGCG assists in enhancing the therapeutic effect. The prepared nanomaterial can achieve controlled release of drugs in the weakly acidic and high ROS environment of diabetic nephropathy lesions, accurately act on the lesion site, improve the therapeutic effect and reduce systemic toxicity. Experimental verification shows that compared with ordinary berberine preparations, the nanomaterial has better effects at the same berberine dose with the same efficacy. After oral administration, the drug concentration in the kidney area is increased and the systemic drug exposure is reduced. In addition, the preparation process is simple, the cost is controllable, and it is both efficient and economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a scanning electron microscope image of the berberine nanomaterial prepared in Example 1; Figure 2 is the expression diagram of apoptosis-related proteins in glomerular endothelial cells; Figure 3 This is the HPLC test chart of berberine nanomaterials and berberine standard solution; Figure 4 This is the test diagram of berberine loading and encapsulation efficiency in berberine nanomaterials; Figure 5 This is a diagram of cell activity under different concentrations of berberine nanomaterials. DETAILED DESCRIPTION

[0017] The specific implementation of the present invention is described in detail below with reference to the embodiments.

[0018] Example 1 A method for preparing a berberine nanomaterial comprises the following steps: S1: Disperse ZIF-8 material in water to obtain a ZIF-8 suspension, then weigh 15% of EGCG (epigallocatechin gallate) and 35% of BBR (berberine) in DMSO (dimethyl sulfoxide) to obtain EGCG solution and BBR solution respectively; S2: EGCG solution and BBR solution were added to the ZIF-8 suspension to obtain a mixed solution, which was then incubated at 37°C and 250 rpm for 7 h, followed by dialysis for 30 h. S3: After centrifuging the dialyzed solution, the precipitate was washed with deionized water, and then the precipitate was freeze-dried at -50°C and a vacuum degree of less than 10 Pa for 28 hours to obtain berberine nanomaterials.

[0019] Example 2 A method for preparing a berberine nanomaterial comprises the following steps: S1: ZIF-67 material is dispersed in water to obtain a ZIF-67 suspension, and then EGCG (epigallocatechin gallate) accounting for 20% of the mass fraction of berberine nanomaterials and BBR (berberine) accounting for 30% of the mass fraction of berberine nanomaterials are weighed and dissolved in DMSO (dimethyl sulfoxide) to obtain EGCG solution and BBR solution respectively; S2: EGCG solution and BBR solution were added to the ZIF-67 suspension to obtain a mixed solution, which was then incubated at 35°C and 300 rpm for 6 h, followed by dialysis for 24 h. S3: After centrifuging the dialyzed solution, the precipitate was washed with deionized water, and then the precipitate was freeze-dried at -50°C and a vacuum degree of less than 10 Pa for 25 hours to obtain berberine nanomaterials.

[0020] Example 3 A method for preparing a berberine nanomaterial comprises the following steps: S1: ZIF-68 material is dispersed in water to obtain a ZIF-68 suspension, and then EGCG (epigallocatechin gallate) accounting for 10% of the mass fraction of berberine nanomaterials and BBR (berberine) accounting for 40% of the mass fraction of berberine nanomaterials are weighed and dissolved in DMSO (dimethyl sulfoxide) to obtain EGCG solution and BBR solution respectively; S2: EGCG solution and BBR solution were added to the ZIF-68 suspension to obtain a mixed solution, which was then incubated at 40°C and 200 rpm for 8 h, followed by dialysis for 36 h. S3: After centrifuging the dialyzed solution, the precipitate was washed with deionized water, and then the precipitate was freeze-dried at -50°C and a vacuum degree of less than 10 Pa for 30 hours to obtain berberine nanomaterials.

[0021] The berberine nanomaterials prepared in Examples 1 to 3 have similar performance and effects, and the berberine nanomaterial prepared in Example 1 was used in subsequent experiments.

[0022] Experimental Example 1 Microscopic analysis: The berberine nanomaterial prepared in Example 1 was analyzed using a scanning electron microscope. The results are as follows: Figure 1 As shown in the figure, it intuitively shows that epigallocatechin gallate (EGCG) and berberine (BBR) are loaded into the pores of the ZIF framework through physical adsorption and chemical bonding using metal organic framework material (ZIF) as a carrier.

[0023] Experimental Example 2 Animal Model Preparation: db / db mice and C57BLKS mice were selected. The model mice were randomly divided into four groups, with six mice in each group. The normal control group received no treatment, the model control group received a solvent by gavage, the normal control group received a 100 mg / kg berberine solution by gavage, and the experimental group received a berberine nanomaterial (ZIF-EGCG-BBR nanomaterial) solution containing a 100 mg / kg berberine solution with equivalent efficacy. The solvent used in all groups was normal saline.

[0024] The drug was administered by gavage once daily for 6 weeks. Body weight and fasting blood glucose levels were measured. After administration, the mice were sacrificed, and blood, urine, and kidney tissue were collected.

[0025] Experimental Example 3 Effect of apoptosis-related protein expression on glomerular endothelial cells: The glomerular endothelial cells in the kidney tissue of the mice in the above-mentioned experimental example 1 were detected, and the results were as follows: Figure 2 As shown, compared with the normal control group, model control group and common control group, the expression of Caspase-3 protein in the experimental group was significantly reduced, and the expression of tubulin protein had a similar effect, which indicates that the berberine nanomaterial of the present invention can more effectively inhibit the apoptosis of glomerular endothelial cells.

[0026] Experimental Example 4 Berberine drug loading detection: The berberine drug loading detection was performed using high performance liquid chromatography (HPLC). First, a berberine standard solution in the range of 5–100 μg / mL was prepared, its peak area was measured, and a standard curve (R2 =0.9992). Subsequently, a certain amount of the berberine nanomaterial prepared in Example 1 (e.g., 1 mg) was weighed and added to PBS buffer and shaken for 2 hours to fully release the berberine therein. The unencapsulated free berberine was removed by a 10 kDa ultrafiltration centrifuge tube, and the filtrate was collected and the berberine content therein was detected by HPLC ( Figure 3 ), combined with the initial input amount to calculate the drug loading (DL%) and encapsulation efficiency (EE%). The experimental results are as follows Figure 4 , the drug loading capacity of the nanomaterial was about 8.6±0.4%, and the encapsulation efficiency was about 82.3±2.7%, indicating that the prepared nanomaterial had good drug loading capacity and encapsulation efficiency.

[0027] Experimental Example 5 Cytotoxicity experiment: L929 cell suspension was dispersed in a 96-well microplate with a volume of 100 μL / well and cultured at 37°C for about 48 hours until it was in the exponential growth phase. Then, the culture medium was removed and supplemented with 100 μL of culture medium containing different concentrations of berberine nanomaterials (7.5 μM, 15 μM, 30 μM, 60 μM, 90 μM and 120 μM) for 24 hours to test cell viability. The results are as follows: Figure 5 As shown, when the concentration of berberine nanomaterials is 30uM and below, the cell viability is maintained above 80%, indicating that the nanomaterials of the present invention have no obvious toxicity to cells within a wide concentration range and have good biosafety.

[0028] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing berberine nanomaterial, characterized in that: The following steps are involved: S1: Disperse the ZIF material in water to obtain a ZIF suspension, and then dissolve EGCG and BBR in DMSO to obtain EGCG solution and BBR solution respectively; S2: Add EGCG solution and BBR solution to the ZIF suspension to obtain a mixed solution, then incubate the mixed solution at 35-40°C and 200-300 rpm for 6-8 hours, followed by dialysis for 24-36 hours. S3: The dialyzed solution is centrifuged, the precipitate is washed, and the precipitate is freeze-dried to obtain berberine nanomaterials.

2. The method for preparing the berberine nanomaterial according to claim 1, wherein: The ZIF material is ZIF-8, ZIF-67 or ZIF-68.

3. The method for preparing the berberine nanomaterial according to claim 1, wherein: The freeze drying is carried out at -50°C and a vacuum degree lower than 10 Pa for 25 to 30 hours.

4. The berberine nanomaterial prepared by the method according to any one of claims 1 to 3.

5. The method for preparing the berberine nanomaterial according to claim 4, wherein: The mass fraction of EGCG in the berberine nanomaterial is 10-20%.

6. The method for preparing the berberine nanomaterial according to claim 5, wherein: The mass fraction of BBR in the berberine nanomaterial is 30-40%.

7. Use of the berberine nanomaterial according to any one of claims 4 to 6 in the preparation of kidney-targeted drugs.

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