Preparation method of asiaticoside and berberine co-loaded PLGA (poly (lactic-co-glycolic acid)) nanoparticles

Abstract

The invention discloses a preparation method of asiaticoside and berberine co-loaded PLGA (poly (lactic-co-glycolic acid)) nanoparticles, which comprises the following steps: weighing asiaticoside, berberine and PLGA, putting into an EP (epoxy resin) tube, adding a methanol and ethanol mixed system, and completely dissolving to obtain a solution I; preparing a PVA solution with the volume fraction of 1%; the first solution is magnetically stirred at the room temperature, the stirring frequency is 120 r / min, and a second solution is obtained; sucking a PVA solution by using an injector, and slowly dripping the PVA solution into the solution II obtained in the step 3 to obtain a solution III; stirring the solution III, and performing ultrasonic treatment in an ice bath; after the ultrasonic treatment is finished, purifying by using an ultrafiltration tube to remove free drugs and methanol and ethanol solutions, so as to obtain asiaticoside and berberine co-loaded PLGA (poly (lactic-co-glycolic acid) nanoparticles; according to the invention, the solubility and the stability of the raw material medicines are improved, so that the common encapsulation efficiency of Asi and Ber is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine. Background Technology

[0002] Polylactic acid-glycolic acid copolymer (PLGA) is a biodegradable synthetic polymer material that has become an ideal carrier for nanomedicine delivery systems due to its good biocompatibility, tunable degradation rate, and sustained drug release capability. In recent years, research on PLGA nanoparticles targeting natural anti-inflammatory active ingredients has gradually increased. Asiaticoside (Asi) exerts its anti-inflammatory effects by inhibiting multiple pathways, including NF-κB, HO-1, and miR-155 / SOCS1, while berberine (Ber) alleviates sepsis-related damage by regulating the NLRP3 inflammasome and purinergic pathways. The combination of these two has synergistic anti-inflammatory potential, but due to differences in their physicochemical properties (such as solubility and molecular polarity) and metabolic mismatch in vivo, direct oral administration suffers from low bioavailability and poor targeting. PLGA nanoparticle co-loading technology can provide a new strategy to address these problems by encapsulating the two drugs, controlling release behavior, and enhancing targeting.

[0003] Current research has successfully loaded Asi or Ber alone into PLGA nanoparticles. For example, the emulsion solvent evaporation method was used to prepare asiaticoside-PLGA nanoparticles with an encapsulation efficiency of over 70%; berberine was released via nanoprecipitation. Single-drug loading processes are simple and parameters, such as solvent selection, can be optimized based on drug properties. Existing dual-drug co-loaded PLGA nanoparticles are mostly for drugs with similar physicochemical properties, such as hydrophobic-hydrophobic combinations, achieved through double emulsion or microfluidic technology. For example, the curcumin and quercetin co-loaded system achieves simultaneous release by adjusting the molecular weight of PLGA. However, dual-loaded PLGA nanoparticles also have some limitations, such as differences in encapsulation efficiency and poor process compatibility. The solubility difference between asiaticoside and berberine makes it difficult to achieve efficient encapsulation using traditional single methods, with dual-drug encapsulation efficiency <50%. Solvent selection must consider both PLGA solubility and the stability of both drugs; for example, asiaticoside easily precipitates in acetone. Current technologies suffer from low and unstable encapsulation rates of PLGA nanoparticles loaded with Asi and Ber, primarily due to the significant difference in solubility between the two drugs: asiaticoside is highly water-soluble, while berberine is highly lipid-soluble, making it difficult to dissolve them simultaneously in traditional solvent systems (such as dichloromethane / water). Furthermore, large-scale production of dual-loaded systems is challenging due to the cumbersome double emulsion process, the high cost of microfluidic equipment, and the need for strict quality control in dual-drug loading. Therefore, we need to improve existing processes to prevent nanoparticle aggregation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine, which can effectively solve the technical problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine includes the following steps: Step 1: Weigh out asiaticoside, berberine, and PLGA in a mass ratio of (1~2):(1~2):(10~15), place them in an EP tube, add 10 mL of a methanol and ethanol mixture, and dissolve completely to obtain solution one; Step 2: Prepare a 1% (v / v) PVA solution; Step 3: Stir the solution from Step 1 magnetically at room temperature at a stirring frequency of 120 r / min to obtain solution 2. Step 4: Using a 1 mL syringe, slowly add 1% PVA solution dropwise into Solution 2 from Step 3 above, for a total of 5 mL of 1% PVA solution, to obtain Solution 3; Step 5: After stirring the solution from Step 4 three times, place it in an ice bath and sonicate for 15 minutes under the following conditions: 3 seconds on, 2 seconds off, and 180W power. Step 6: After sonication, the free drug, methanol and ethanol solution were removed by purifying with a 100 kDa ultrafiltration tube to obtain PLGA nanoparticles co-loaded with asiaticoside and berberine. The particle size of PLGA@Asi / Ber indicates the PLGA nanoparticles co-loaded with asiaticoside and berberine. The free drug includes unencapsulated small molecule drugs of asiaticoside and berberine. Step 7: Detect the Asi content of the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using high performance liquid chromatography (HPLC); Step 8: Determine the content of Ber in the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using a UV spectrophotometer; Step 9: The particle size and morphology of Asi, which is co-loaded with PLGA nanoparticles by asiaticoside and berberine in Step 6, were detected by Malvern particle size analyzer (DLS) and transmission electron microscopy (TEM). Step 10: The toxicity of PLGA nanoparticles co-loaded with asiaticoside and berberine was determined using RAW264.7 macrophages and HUVEC cells.

[0006] Preferably, in step 1, asiaticoside is first dissolved in 4 mL of ethanol, then berberine and PLGA are dissolved in 6 mL of methanol, and finally the mixture is ultrasonically mixed for 5 min.

[0007] Preferably, in step 6, the centrifugation speed is 3000 rpm / min and the centrifugation time is 15~30 min.

[0008] Preferably, in step 6, the ultrafiltration is performed at least 3 times, and the ultrafiltration temperature is 4°C.

[0009] Preferably, the liquid phase conditions for Asi are: acetonitrile to β-cyclodextrin volume ratio of 24:76, wavelength of 210 nm, column temperature of 30℃, C18 column of 5 μm, 4.6*250 mm, and β-cyclodextrin concentration of 2 mmol / L.

[0010] Preferably, in step 8, the detection wavelength of Ber is 350 nm.

[0011] Preferably, in step 4, 1% PVA by volume is slowly added dropwise to solution 2 at a rate of one drop per second, and the mixture is stirred for 3-5 minutes after addition.

[0012] Preferably, the PLGA has a molecular weight of 10,000-20,000 and a molar ratio of lactic acid to hydroxylactic acid of 50:50.

[0013] Preferably, in step 1, 1 mg of asiaticoside, 1 mg of berberine, and 10 mg of PLGA are weighed out.

[0014] Application of PLGA nanoparticles co-loaded with asiaticoside and berberine prepared by the above preparation method in promoting tissue regeneration.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention improves the solubility and stability of drugs and is beneficial for increasing the co-encapsulation efficiency of Asi and Ber. The method of this invention also utilizes the principle of ultrasonic emulsification and solvent evaporation, but by removing surfactant components and changing the addition method, including slow PVA dripping, and increasing emulsification time, the co-encapsulation efficiency and drug loading of Asi and Ber are increased. Furthermore, due to the drug properties encapsulated by PLGA nanoparticles according to this invention, using a methanol and ethanol mixture as a solvent better dissolves Asi and Ber, resulting in spherical PLGA co-loaded nanoparticles with good stability and dispersibility. A potential less than -10 mV indicates nanoparticle stability, and a dispersibility coefficient less than 0.3 indicates good dispersibility. This method eliminates the surfactant required in ultrasonic emulsification, making the method simpler and more convenient. Attached Figure Description

[0016] Figure 1 The basic particle size of PLGA@Asi / Ber in this application embodiment; Figure 2The polydispersity index (PDI) of PLGA@Asi / Ber in this application embodiment; Figure 3 The potential of PLGA@Asi / Ber in the embodiments of this application; Figure 4 The image shown is an electron microscope image of PLGA@Asi / Ber from an embodiment of this application. The scale bar is 100 nm. Figure 5 This is a graph showing the Asi content determination of PLGA@Asi / Ber in the embodiments of this application; Figure 6 This is a graph showing the content determination of Ber in PLGA@Asi / Ber in the embodiments of this application; Figure 7 The results of the cytotoxicity experiment of PLGA@Asi / Ber in the embodiments of this application are the experimental results of cytotoxicity of PLGA@Asi / Ber against RAW 264.7 (n=3). Figure 8 The figure shows the experimental results of PLGA@Asi / Ber on the cytotoxicity (n=3) of HUVEC cells in the embodiments of this application. Detailed Implementation

[0017] The technical solution of the present invention will be described in detail below with reference to the embodiments, but the scope of protection is not limited thereto. Example 1

[0018] A method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine, specifically including the following steps: Step 1: Weigh 1 mg of asiaticoside, 1 mg of berberine, and 10 mg of PLGA into an EP tube, add 10 mL of a methanol and ethanol mixture, and dissolve completely to obtain solution one; Step 2: Prepare a 1% (v / v) PVA solution; Step 3: Stir the solution from Step 1 magnetically at room temperature at a stirring frequency of 120 r / min to obtain solution 2. Step 4: Using a 1 mL syringe, slowly add 1% PVA solution dropwise into Solution 2 from Step 3 above, for a total of 5 mL of 1% PVA solution, to obtain Solution 3; Step 5: After stirring the solution from Step 4 three times, place it in an ice bath and sonicate for 15 minutes under the following conditions: 3 seconds on, 2 seconds off, and 180W power. Step 6: After sonication, the free drug, methanol and ethanol solution were removed by purifying with a 100 kDa ultrafiltration tube to obtain PLGA nanoparticles co-loaded with asiaticoside and berberine. The particle size of PLGA@Asi / Ber indicates the PLGA nanoparticles co-loaded with asiaticoside and berberine. Step 7: Detect the Asi content of the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using high performance liquid chromatography (HPLC); Step 8: Determine the Ber content of Asi in the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using a UV spectrophotometer. Step 9: The particle size and morphology of Asi, which is co-loaded with PLGA nanoparticles by asiaticoside and berberine in Step 6, were detected by Malvern particle size analyzer (DLS) and transmission electron microscopy (TEM). Step 10: Assess cytotoxicity using RAW264.7 macrophages and HUVEC cells. Example 2

[0019] A method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine, specifically including the following steps: Step 1: Weigh 2 mg of asiaticoside, 2 mg of berberine, and 15 mg of PLGA into an EP tube. The molecular weight of PLGA is 10,000-20,000, and the molar ratio of lactic acid to hydroxyl lactic acid is 50:50. First, dissolve asiaticoside in 4 mL of ethanol, then dissolve berberine and PLGA in 6 mL of methanol. Finally, mix by sonication for 5 min until complete dissolution to obtain solution one. Step 2: Prepare a 1% (v / v) PVA solution; Step 3: Stir the solution from Step 1 magnetically at room temperature at a stirring frequency of 120 r / min to obtain solution 2. Step 4: Using a 1 mL syringe, slowly add 1% PVA solution dropwise into solution two from step 3 above, for a total of 5 mL of 1% PVA solution. The speed should be controlled at one drop per second. Stir for 3-5 minutes after adding the solution to obtain solution three. Step 5: After stirring the solution from Step 4 three times, place it in an ice bath and sonicate for 15 minutes under the following conditions: 3 seconds on, 2 seconds off, and 180W power. Step 6: After sonication, the free drug, methanol and ethanol solution were removed by purifying with a 100 kDa ultrafiltration tube. The centrifugation speed was 3000 rpm / min and the centrifugation time was 15 min. The ultrafiltration was performed at least 3 times and the ultrafiltration temperature was 4℃ to obtain PLGA nanoparticles co-loaded with asiaticoside and berberine. Step 7: The content of Asi in the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 was detected by high performance liquid chromatography (HPLC). The liquid chromatography conditions for Asi were: acetonitrile to β-cyclodextrin volume ratio of 24:76, wavelength of 210 nm, column temperature of 30℃, C18 column of 5 μm and 4.6*250 mm, and β-cyclodextrin concentration of 2 mmol / L. Step 8: Determine the Ber content of Asi in the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using a UV spectrophotometer. The detection wavelength of Ber is 350 nm. Step 9: The particle size and morphology of Asi, which is co-loaded with PLGA nanoparticles by asiaticoside and berberine in Step 6, were detected by Malvern particle size analyzer (DLS) and transmission electron microscopy (TEM). Step 10: Assess cytotoxicity using RAW264.7 macrophages and HUVEC cells. Example 3

[0020] A method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine, specifically including the following steps: Step 1: Weigh 1 mg of asiaticoside, 1 mg of berberine, and 10 mg of PLGA into an EP tube. The molecular weight of PLGA is 10,000-20,000, and the molar ratio of lactic acid to hydroxyl lactic acid is 50:50. First, dissolve asiaticoside in 4 mL of ethanol, then dissolve berberine and PLGA in 6 mL of methanol. Finally, mix by sonication for 5 min until complete dissolution to obtain solution one. Step 2: Prepare a 1% (v / v) PVA solution; Step 3: Stir the solution from Step 1 magnetically at room temperature at a stirring frequency of 120 r / min to obtain solution 2. Step 4: Using a 1 mL syringe, slowly add 1% PVA solution dropwise into solution two from step 3 above, for a total of 5 mL of 1% PVA solution. The speed should be controlled at one drop per second. Stir for 3-5 minutes after adding the solution to obtain solution three. Step 5: After stirring the solution from Step 4 three times, place it in an ice bath and sonicate for 15 minutes under the following conditions: 3 seconds on, 2 seconds off, and 180W power. Step 6: After sonication, the free drug, methanol and ethanol solution were removed by purifying with a 100 kDa ultrafiltration tube. The centrifugation speed was 3000 rpm / min and the centrifugation time was 30 min. The ultrafiltration was performed at least 3 times and the ultrafiltration temperature was 4℃ to obtain PLGA nanoparticles co-loaded with asiaticoside and berberine. Step 7: The content of Asi in the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 was determined by high performance liquid chromatography (HPLC). The liquid chromatography conditions for Asi were acetonitrile:β-cyclodextrin = 24:76, wavelength 210 nm, column temperature 30℃, C18 column 5 μm, 4.6*250 mm, and β-cyclodextrin concentration 2 mmol / L. Step 8: Determine the Ber content of Asi in the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using a UV spectrophotometer. The detection wavelength of Ber is 350 nm. Step 9: The particle size and morphology of Asi, which is co-loaded with PLGA nanoparticles by asiaticoside and berberine in Step 6, were detected by Malvern particle size analyzer (DLS) and transmission electron microscopy (TEM). Step 10: Assess cytotoxicity using RAW264.7 macrophages and HUVEC cells.

[0021] Prepare Asi standards at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 μg / mL. Centrifuge at 12000 rpm for 15 min, and inject the supernatant as a standard solution into the chromatographic column. An autosampler or manual injector is typically used. Turn on the HPLC instrument and start column operation. As the solution passes through the column, the components in the sample will gradually separate and pass through the detector. The detector is used to detect the sample components, and chromatograms and peak areas are collected. Take 200 μL of PLGA@Asi / Ber nanoparticles from Example 1 of this invention, add 800 μL of methanol, and ultrasonically demulsify for 30 min using an ultrasonic cleaner. After demulsification, centrifuge the sample at 12000 rpm for 20 min, collect the supernatant, load it onto the column, and record the peak area data under HPLC conditions.

[0022] The experimental method for determining the content of Berber using a UV-Vis spectrophotometer involves preparing Berber standard solutions with methanol at concentrations of 20, 10, 5, 2.5, 1.25, and 0.625 μg / mL. The absorbance of each Berber standard solution at different concentrations is measured using a UV-Vis spectrophotometer. A standard curve is obtained by plotting concentration on the x-axis and absorbance on the y-axis, as shown below. Figure 6Take 200 μL of the PLGA@Asi / Ber nanoparticles of this invention, add 800 μL of methanol, and ultrasonically demulsify for 30 min using an ultrasonic cleaner. After demulsification, centrifuge the sample at 12000 rpm for 20 min at 4℃, and take the supernatant for UV absorption measurement. Turn on the UV spectrophotometer and preheat the equipment for 10-15 minutes. First, perform zero-point calibration by adding pure methanol to the photocell, selecting the zero-point calibration mode, adjusting the wavelength to the zero-point value, and then pressing the zero-point calibration button. Transfer the sample to the photocell and adjust the wavelength to 350 nm. Between nm, click the "Measure" button, record the absorbance value, and after completing the test, clean the optical cell with pure solvent and turn off the power of the device; measure the absorbance value y1 of an unknown concentration of the PLGA@Asi / Ber demulsified sample prepared in Example 1 of this invention under a UV-Vis spectrophotometer, substitute y1 into the standard curve of Ber to obtain the corresponding X1, and thus determine the specific concentration value of Ber in PLGA@Asi / Ber nanoparticles, and calculate the drug encapsulation efficiency and drug loading. Particle size and PDI (polydispersity index) determination: After diluting the PLGA@Asi / Ber prepared in Example 1 of this invention with ultrapure water, the particle size, polydispersity index (PDI) and potential of PLGA@Asi / Ber were measured using a Malvern particle size potentiometer. Transmission electron microscope (TEM) imaging: A copper grid was placed on adhesive paper. The PLGA@Asi / Ber prepared in Example 1 of this invention was fixed with glutaraldehyde. Then, 5-10 μL of the PLGA@Asi / Ber solution prepared in Example 1 of this invention was added to the copper grid and allowed to stand for 10 min (waiting for adsorption). Then, excess sample on the copper grid was removed with filter paper. Then, 10 μL of 2% phosphotungstic acid staining solution was added to the copper grid and stained for 2 min. Excess staining solution was carefully removed with filter paper. The copper grid was dried under an infrared lamp. Finally, the morphology of EVs was observed using a transmission electron microscope. Cytotoxicity assay: The toxic effects of different concentrations of PLGA@Asi / Ber (according to Ber concentration) from Example 1 of this invention on RAW264.7 and HUVEC were studied using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) method. HUVECs and RAW264.7 in logarithmic growth phase were digested and then subjected to 8 × 10⁻⁶ cytotoxicity assays. 3Cells were seeded at a density of 100 μL of cell suspension in each well of a 96-well plate and allowed to adhere. Once the cells reached 80% confluence, 100 μL of different concentrations of PLGA@Asi / Ber (according to the Ber concentration) were added, with Ber concentrations of 0, 3.75, 7.5, 15, 30, and 60 μg / mL. After incubating the cells with the drug for 48 h, the drug solution was removed, and 100 μL of 1 mg / mL MTT solution was added. After incubating for 4 h, the MTT solution was removed, and 100 μL of DMSO was added to each well. The plate was then shaken at room temperature for 15 min until the formazan was completely dissolved. The absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated. Experimental results: The particle size detected by the laser diffractometer (NANO ZS90) was 171.17 ± 1.03 nm, the PDI was 0.12 ± 0.02, and the potential was -14.43 ± 3.57 mV (e.g., Figure 1-3 );from Figure 4 As can be seen from the electron microscopy morphology, PLGA@Asi / Ber is spherical with good dispersibility and a particle size of about 100 nm. Figure 5 The standard curve of Asi in PLGA@Asi / Ber is shown. Calculations show that the drug loading of Asi in PLGA@Asi / Ber is 4.4%. Figure 6 The UV standard curve for Ber was used, and the drug loading of Ber in PLGA@Asi / Ber was calculated to be 2.4%. According to the data in Table 1, the drug loading and encapsulation efficiency of the two drugs in the system can be obtained, indicating successful preparation. In this preparation method, the mass ratio of Asi to Ber is 1:1, and the mass ratio of Asi to Ber in the obtained sample is 2:1. (The last sentence appears to be incomplete and possibly refers to a separate process involving PLGA@Asi / Ber.) Figure 7 and Figure 8 The study found that PLGA@Asi / Ber, with Ber concentrations between 0 and 60 μg / mL, did not cause cytotoxicity in RAW264.7 and HUVEC cells, indicating that the invention has good safety. In particular, when the Ber concentration reached 15 μg / mL in HUVEC cells, it promoted HUVEC proliferation. Since the Asi concentration at this point was 30 μg / mL, it could stimulate fibroblast proliferation. These data indicate that the invention has the potential to promote tissue regeneration.

[0023] Table 1 shows the drug loading and encapsulation efficiency data of the two drugs in PLGA@Asi / Ber of this application. Under the same experimental conditions, the samples in Examples 1, 2 and 3 of this invention were used to conduct repeat experiments to obtain the first, second and third sets of data in Table 1, respectively, in order to increase the authenticity and stability of the data. Table 1 Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine, characterized in that, Includes the following steps, Step 1: Weigh out asiaticoside, berberine, and PLGA in a mass ratio of (1-2):(1-2):(10-15), place them in an EP tube, add 10 mL of a methanol and ethanol mixture, and dissolve completely to obtain solution one; Step 2: Prepare a 1% (v / v) PVA solution; Step 3: Stir the solution from Step 1 magnetically at room temperature at a stirring frequency of 120 r / min to obtain solution 2. Step 4: Using a 1mL syringe, slowly add 1% PVA solution dropwise into solution two from step 3 above, for a total of 5mL of 1% PVA solution, to obtain solution three; Step 5: After stirring the solution from Step 4 three times, place it in an ice bath and sonicate for 15 minutes under the following conditions: 3 seconds on, 2 seconds off, and 180W power. Step 6: After sonication, the free drug, methanol and ethanol solution were removed by purifying with a 100kDa ultrafiltration tube to obtain PLGA nanoparticles co-loaded with asiaticoside and berberine. The free drug included unencapsulated asiaticoside and berberine small molecule drugs. Step 7: Detect the Asi content of the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using high performance liquid chromatography; Step 8: Determine the content of Ber in the PLGA nanoparticles co-loaded with asiaticoside and berberine in Step 6 using a UV spectrophotometer; Step 9: The particle size and morphology of Asi, which is co-loaded with PLGA nanoparticles by asiaticoside and berberine in Step 6, were detected by Malvern particle size analyzer and transmission electron microscopy. Step 10: The toxicity of PLGA nanoparticles co-loaded with asiaticoside and berberine was determined using RAW264.7 macrophages and HUVEC cells.

2. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, characterized in that, In step 1, asiaticoside is first dissolved in 4 mL of ethanol, then berberine and PLGA are dissolved in 6 mL of methanol, and finally the mixture is ultrasonically mixed for 5 min.

3. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, characterized in that, In step 6, the centrifugation speed is 3000 rpm / min and the centrifugation time is 15 to 30 min.

4. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, characterized in that, In step 6, ultrafiltration is performed at least three times at a temperature of 4°C.

5. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, wherein in step 7, the liquid phase conditions of Asi are acetonitrile:β-cyclodextrin = 24:76, wavelength is 210 nm, column temperature is 30 °C, C18 column is 5 μm, 4.6*250 mm, and β-cyclodextrin concentration is 2 mmol / L.

6. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, characterized in that, In step 8, the detection wavelength for Ber is 350 nm.

7. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, characterized in that, In step 4, 1% PVA by volume is slowly added dropwise to solution 2 at a rate of one drop per second, and then stirred for 3-5 minutes.

8. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, characterized in that, The PLGA has a molecular weight of 10,000-20,000 and a lactic acid:hydroxyl lactic acid molar ratio of 50:

50.

9. The method for preparing PLGA nanoparticles co-supported with asiaticoside and berberine according to claim 1, characterized in that, In step 1, 1 mg of asiaticoside, 1 mg of berberine, and 10 mg of PLGA were weighed out.

10. The application of PLGA nanoparticles co-loaded with asiaticoside and berberine prepared according to any one of claims 1-9 in promoting tissue regeneration.

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