Homogeneous polysaccharide-stabilized puerarin nanoribbons and a preparation method thereof

Abstract

The invention discloses a homologous polysaccharide-stabilized puerarin nanobelt and a preparation method thereof, and belongs to the technical field of drug nanobelt. The puerarin nanobelt is prepared from puerarin and pueraria polysaccharide by a medium grinding method. The preparation method comprises the following steps: weighing puerarin raw medicine and pueraria polysaccharide raw medicine; adding the pueraria polysaccharide into water and dissolving completely; then adding the puerarin, a stirring rod and 0.4-0.6 mm zirconium oxide beads with the same volume as the water; placing a vial on a magnetic stirrer and grinding at a magnetic stirring speed of 800-1400 rpm for 2-12 h; taking out, filtering and removing the zirconium oxide beads to obtain the puerarin nanobelt. The invention has the following advantages: the polysaccharide stabilizer is of natural origin, has good biocompatibility, low toxicity and biodegradability, and has significant advantages in the nanometer delivery system; the cumulative dissolution amount is greatly increased, and the equilibrium time is greatly shortened; and the oral absorption of puerarin can be significantly improved.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceutical nanoribbons (pharmaceutical preparations characterized by special physical shapes), specifically relating to a homologous polysaccharide-stabilized puerarin nanoribbon and its preparation method. Background Technology

[0002] Kudzu root, the root of the legume *Pueraria lobata* or *Pueraria truncata*, contains abundant bioactive components and possesses a wide range of pharmacological effects. Puerarin (PRN), one of its main active components, exhibits various biological activities, including anti-inflammatory, anticancer, and cardiovascular protective effects. However, PRN's low solubility in water and limited bioavailability significantly restrict its further clinical application. To address this deficiency, researchers have proposed various strategies, among which nanotechnology has attracted considerable attention due to its ability to significantly improve the solubility and bioavailability of poorly soluble drugs.

[0003] Currently, the preparation of nano-formulations often relies on chemically synthesized stabilizers (such as sodium dodecyl sulfate and Tween 80), but long-term use of these excipients may pose potential safety hazards. In contrast, naturally derived stabilizers such as polysaccharides, phospholipids, and saponins exhibit significant advantages in nanodelivery systems due to their good biocompatibility, low toxicity, and biodegradability. Summary of the Invention

[0004] Pueraria polysaccharide (PPL), as a natural polysaccharide, not only has antioxidant and other biological activities, but may also have a synergistic effect with its homologous puerarin in pharmacology. At the same time, it can be used as a stabilizer to improve the stability and safety of nano-formulations.

[0005] Therefore, this invention proposes a preparation strategy for puerarin nanoribbons (PRN NB) using PPL as a stabilizer. By optimizing the formulation and process parameters through media milling, structurally stable and uniformly dispersed homologous polysaccharide-stabilized puerarin nanoribbons were prepared, and their physicochemical properties, stability, in vitro release behavior, and pharmacokinetic properties were systematically evaluated. This research aims to develop a green, efficient, and biocompatible nanodelivery system, providing a new formulation option for the clinical application of puerarin.

[0006] The first objective of this invention is to disclose a homopolysaccharide-stabilized puerarin nanoribbon.

[0007] The second objective of this invention is to disclose the preparation method of the above-mentioned puerarin nanoribbons.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A homologous polysaccharide-stabilized puerarin nanoribbon, wherein the puerarin nanoribbon is prepared from puerarin and puerarin polysaccharide by a media milling method.

[0010] The puerarin nanoribbons described in the above technical solution are prepared by the following steps:

[0011] (1) Weigh out puerarin raw material and puerarin polysaccharide raw material according to the mass ratio w / w between puerarin raw material and puerarin polysaccharide raw material of 10:2 ~ 10:12, and set aside for later use;

[0012] (2) Add kudzu polysaccharide to a vial, add water, and after it is completely dissolved, obtain a solution with a kudzu polysaccharide concentration of 0.5~1.5% (w / v);

[0013] (3) Add puerarin to step (2) so that the concentration of puerarin in the solution is 0.5 ~ 1.5% (w / v); then add a stir bar and 0.4 ~ 0.6 mm zirconium oxide beads of the same volume as water, place the vial on a magnetic stirrer, and grind at a magnetic stirring speed of 800 ~ 1400 rpm for 2 ~ 12 h, take it out, filter to remove the zirconium oxide beads, and obtain puerarin nanoribbons.

[0014] The puerarin nanoribbons described in the above technical solution, wherein the steps are as follows:

[0015] (1) Weigh 40 mg of puerarin raw material and 40 mg of puerarin polysaccharide for later use;

[0016] (2) Add kudzu polysaccharide to a 10 mL vial and add 4 mL of water; after it is completely dissolved, a solution with a kudzu polysaccharide concentration of 1% (w / v) is obtained;

[0017] (3) Add 40 mg of puerarin to step (2) to make the concentration of puerarin in the solution 1% (w / v), then add a stir bar and 4 mL of 0.4 ~ 0.6 mm zirconia beads. Place the vial on a magnetic stirrer, set the magnetic stirring speed to 1000 rpm and the grinding time to 4 h, take it out, filter to remove the zirconia beads, and obtain puerarin nanoribbons.

[0018] The puerarin nanoribbons described in the above technical solution have a particle size of (174.13 ± 8.11) nm, a polydispersity index of (0.2057 ± 0.0185), and a zeta potential of (-15.29 ± 0.96) mV.

[0019] The puerarin nanoribbons described in the above technical solution are wherein the puerarin nanoribbon dispersion is ribbon-shaped, with a width of 50~100 nm and a length of 0.5~2 μm.

[0020] The puerarin nanoribbons described in the above technical solution have a drug loading of (46.95 ± 0.52)%.

[0021] The puerarin nanoribbons described in the above technical solution, wherein the puerarin nanoribbons cumulatively dissolves 85.57% in simulated intestinal fluid over 60 minutes.

[0022] A method for preparing homologous polysaccharide-stabilized puerarin nanoribbons includes the following steps:

[0023] (1) Weigh out puerarin raw material and puerarin polysaccharide raw material according to the mass ratio w / w between puerarin raw material and puerarin polysaccharide raw material of 10:2 ~ 10:12, and set aside for later use;

[0024] (2) Add kudzu polysaccharide to a vial, add water, and after it is completely dissolved, obtain a solution with a kudzu polysaccharide concentration of 0.5~1.5% (w / v);

[0025] (3) Add puerarin to step (2) to make the concentration of puerarin in the solution 0.5 ~ 1.5% (w / v), then add a stir bar and 0.4 ~ 0.6 mm zirconium oxide beads of the same volume as water. Place the vial on a magnetic stirrer and grind at a magnetic stirring speed of 800 ~ 1400 rpm for 2 ~ 12 h. Take it out, filter to remove the zirconium oxide beads, and you will get puerarin nanoribbons.

[0026] The preparation method described in the above technical solution, wherein the step is as follows:

[0027] (1) Weigh 40 mg of puerarin raw material and 40 mg of puerarin polysaccharide for later use;

[0028] (2) Add kudzu polysaccharide to a 10 mL vial and add 4 mL of water; after it is completely dissolved, a solution with a kudzu polysaccharide concentration of 1% (w / v) is obtained;

[0029] (3) Add 40 mg of puerarin to step (2) to make the concentration of puerarin in the solution 1% (w / v), then add a stir bar and 4 mL of 0.4 ~ 0.6 mm zirconia beads. Place the vial on a magnetic stirrer, set the magnetic stirring speed to 1000 rpm and the grinding time to 4 h, take it out, filter to remove the zirconia beads, and obtain puerarin nanoribbons.

[0030] The present invention has the following beneficial effects:

[0031] 1. The puerarin nanoribbons (PRN NB) of the present invention uses a naturally derived polysaccharide stabilizer, which has better biocompatibility, low toxicity and biodegradability than chemically synthesized stabilizers, and shows significant advantages in nanodelivery systems.

[0032] 2. The equilibrium solubility of the PRN NB of the present invention is 2.33 times greater than that of PRN in water.

[0033] 3. The PRN NB of the present invention achieved a cumulative dissolution of 85.57% in simulated intestinal fluid over 60 minutes, while the cumulative dissolution of PRN active pharmaceutical ingredient was only 30.62%. Compared with PRN active pharmaceutical ingredient, the cumulative dissolution of PRN NB is significantly increased, and the equilibrium time is significantly shortened.

[0034] 4. Compared with PRN raw materials, PRN NB has a higher C content. max and AUC 0-t The increases were 2.01 times and 1.97 times respectively, indicating that preparing PRN into NB significantly improved the oral absorption of PRN. P <0.05). Attached Figure Description

[0035] Figure 1 The effect of different grinding times on the PS and PDI of PRN NB;

[0036] Figure 2 The effect of different stirring speeds on the PS and PDI of PRN NB;

[0037] Figure 3 The effect of different drug-to-stabilizer ratios on PS and PDI of PRN NB;

[0038] Figure 4 Effects of different drug concentrations on PS and PDI of PRN NB;

[0039] Figure 5 PS (A) and Zeta potential diagrams (B) for PRN NB;

[0040] Figure 6 The images are SEM and TEM images; in the SEM image, A is PRN active pharmaceutical ingredient (scale bar = 100 μm), B is PPL active pharmaceutical ingredient (scale bar = 100 μm), and C is PRN NB lyophilized powder (scale bar = 5 μm); the TEM image is D. PRN NB aqueous dispersion (scale bar = 1 μm).

[0041] Figure 7 XRD diffraction analysis pattern;

[0042] Figure 8 The image shows the HPLC chromatogram of methanol, where A is the test solution, B is the reference solution, and C is the blank methanol solution.

[0043] Figure 9 The equilibrium solubility of PRN NB, where compared to PRN, * P <0.05, ** P <0.01;

[0044] Figure 10 For the stability test of PRN NB, which of the following factors are considered? A. PS; B. PDI; C. Zeta potential; D. Drug loading;

[0045] Figure 11 The image shows a specific HPLC chromatogram of PRN NB in ​​simulated intestinal fluid, where A is the test solution, B is the PRN reference solution, and C is the simulated intestinal fluid.

[0046] Figure 12 The in vitro release curves of PRN NB in ​​simulated gastric fluid (A) and simulated intestinal fluid (B) are shown.

[0047] Figure 13 The chromatograms are specific chromatograms, where A and B are blank plasma, C and D are blank plasma containing puerarin and carbamazepine reference standards, and E and F are plasma samples from rats 0.25 h after oral administration of puerarin preparation.

[0048] Figure 14 The plasma concentration-time curve of the PRN formulation (M ± SD, n = 6). Detailed Implementation

[0049] To facilitate understanding of the technical solution of the present invention, the following detailed description, in conjunction with specific embodiments, further illustrates a homologous polysaccharide-stabilized puerarin nanoribbon and its preparation method.

[0050] Example 1: Preparation of puerarin nanoribbons:

[0051] Weigh out 40 mg of PRN raw material and 40 mg of PPL, and set aside. Add PPL to a 10 mL vial, add 4 mL of water, and after it is completely dissolved, add 40 mg of PRN raw material, then add a stir bar and 4 mL of 0.4 ~ 0.6 mm zirconia beads. Place the vial on a magnetic stirrer, set the magnetic stirring speed to 1000 rpm, and the grinding time to 4 h. Remove the vial, filter to remove the zirconia beads, and obtain PRN NB.

[0052] The following specific experimental examples illustrate the preparation process of the present invention and its beneficial effects.

[0053] Experimental Example 1: Preparation process and characterization of puerarin nanoribbons:

[0054] 1.1 Preparation process of puerarin nanoribbons (PRN NB):

[0055] PRN NB was prepared by media grinding. Appropriate amounts of PRN raw material and PPL were weighed and set aside. PPL was added to a 10 mL vial, followed by 4 mL of water. After complete dissolution, the PRN raw material was added, along with a stir bar and 4 mL of 0.4–0.6 mm zirconia beads. The vial was placed on a magnetic stirrer and ground for a certain time at a specific magnetic stirring speed. The vial was then removed, filtered to remove the zirconia beads, and the PRN NB was obtained.

[0056] 1.2 Single-factor optimization of PRN NB formulation and process:

[0057] 1.2.1 Determination of indicators:

[0058] Take an appropriate amount of PRN NB, dilute it with an appropriate amount of purified water, and use a nano-laser particle size analyzer to determine the particle size (PS) and polydispersity index (PDI) of the PRN NB. Repeat the measurement 3 times.

[0059] 1.2.2 Grinding time assessment:

[0060] With a fixed PRN concentration of 1% (w / v) and a PRN to PPL ratio of 10:5 (w / w), and a magnetic stirring speed of 1000 rpm, grinding times of 2, 4, 6, 8, 12, 18, and 24 h were investigated. Each experiment was repeated three times, and the results are shown in the figure. Figure 1 The PS and PDI of PRN NB first decreased and then increased with the extension of grinding time. Compared with the magnetic stirring group for 2 h, the PS of the magnetic stirring groups for 4, 6, 8, and 12 h were significantly different. P The PDI of the magnetically stirred 4 h group was significantly different from that of the magnetically stirred 4 h group (<0.05). Compared with the magnetically stirred 4 h group, there were no significant differences in PS and PDI in the 6, 8, and 12 h groups. To save time, magnetic stirring for 4 h was selected for the next step of the study.

[0061] 1.2.3 Grinding speed assessment:

[0062] With a fixed PRN concentration of 1% (w / v), a PRN to PPL ratio of 10:5 (w / w), and a grinding time of 4 h, the effects of magnetic stirring speeds of 800, 1000, 1200, and 1400 rpm on the PS and PDI of PRN NB were investigated. Each experiment was repeated three times, and the results are shown in the figure. Figure 2The PS and PDI of PRN NB decreased with increasing magnetic stirring speed. Compared with the magnetic stirring group at 800 rpm, the PS and PDI of the 1000, 1200, and 1400 rpm groups showed significant differences. P <0.05), compared with the magnetic stirring 1000 rpm group, there were no significant differences in PS and PDI in the 1200 and 1400 rpm groups. P >0.05). To save energy, a magnetic stirrer at 1000 rpm was selected for the next step of the study.

[0063] 1.2.4 Examination of the ratio of PRN to PPL:

[0064] With a fixed PRN concentration of 1% (w / v), a magnetic stirring speed of 1000 rpm, and a grinding time of 4 h, the effects of PRN to PPL ratios of 10:2, 10:5, 10:6, 10:8, 10:10, and 10:12 (w / w) on the PS and PDI of PRN NB were investigated. Each experiment was repeated three times. The results are shown in […]. Figure 3 The PS and PDI of PRN NB first decreased and then increased with the increase of the stabilizer ratio. Compared with the 10:10 PRN:PPL ratio, the PS of the 10:2 and 10:12 groups was significantly increased. P <0.05), the PDI of the 10:2, 10:5, 10:6, 10:8, and 10:12 groups were all significantly different ( P (<0.05), taking all factors into consideration, a PRN to PPL ratio of 10:10 was selected for further research.

[0065] 1.2.5 Investigation of PRN concentration:

[0066] With a fixed PRN:PPL ratio of 10:10 (w / w), a magnetic stirring speed of 1000 rpm, and a grinding time of 4 h, the effects of PRN concentrations of 0.5%, 0.75%, 1%, 1.25%, and 1.5% (w / v) on the PS and PDI of PRN NB were investigated. Each experiment was repeated three times, and the results are shown in the figure. Figure 4 The PS of PRN NB gradually decreased with increasing drug concentration, but the PDI of PRN NB first decreased and then increased with increasing drug concentration. Compared with the 1% (w / v) PRN dosage, the PS of the 0.5% and 1.5% PRN dosage groups were significantly different. P <0.05), there was no significant difference in PS between the 0.75% and 1.25% (w / v) PRN groups. P >0.05), the PDI was significantly increased in the 1.25% and 1.5% PRN groups ( P <0.05%, considering the PRN dosage, 1% (w / v) was selected.

[0067] 1.2.6 Process Validation:

[0068] The final optimal formulation and process conditions were: PRN concentration of 1% (w / v), PRN:PPL ratio of 10:10 (w / w), magnetic stirring speed of 1000 rpm, and grinding time of 4 h. The specific preparation process is as follows: Weigh 40 mg of PRN raw material and 40 mg of PPL and set aside; add PPL to a 10 mL vial, add 4 mL of water, and after complete dissolution, add 40 mg of PRN raw material, then add a stir bar and 4 mL of 0.4 ~ 0.6 mm zirconia beads. Place the vial on a magnetic stirrer, set the magnetic stirring speed to 1000 rpm, and the grinding time to 4 h. Remove the vial, filter to remove the zirconia beads, and obtain PRN NB.

[0069] 1.2.7 Preparation of PRN NB lyophilized powder:

[0070] PRN NB was pre-frozen at -20℃ and -80℃ for 12 h in sequence, and then dried in a freeze dryer for 24 h to obtain PRN NB freeze-dried powder.

[0071] 1.3 PRN NB characterization:

[0072] 1.3.1 PS distribution and Zeta potential:

[0073] The PRN NB prepared in Example 1 was diluted with an appropriate amount of distilled water. The PS distribution and Zeta potential of the PRN NB were measured using a nanoparticle size potentiostat. The results are shown in [Figure number missing]. Figure 5 The PS of the PRN NB was (174.13 ± 8.11) nm, the PDI was (0.2057 ± 0.0185) mV, and the Zeta potential was (-15.29 ± 0.96) mV.

[0074] 1.3.2 Morphological Analysis:

[0075] PRN active pharmaceutical ingredient, PPL active pharmaceutical ingredient, and PRN NB lyophilized powder were adhered to a silicon substrate, sputtered with gold, and their morphology was observed and photographed under a scanning electron microscope. A drop of the PRN NB dispersion prepared in Example 1 was taken and appropriately diluted and placed on a copper grid; its morphology was observed and photographed under a transmission electron microscope. The results are shown in the figure. Figure 6 As shown in the figure, the PRN raw material is in block form with a particle size of approximately 50-500 μm. Figure 6 A) PPL active pharmaceutical ingredient is irregularly shaped spheres with a particle size of approximately 100~500 μm. Figure 6 B); PRN NB lyophilized powder is in the form of ribbons, 50 ~ 200 nm wide and 0.5 ~ 3 µm long ( Figure 6C); PRN NB dispersions are ribbon-like, 50 ~ 100 nm wide and 0.5 ~ 2 μm long ( Figure 6 D).

[0076] 1.3.3 Crystal form analysis:

[0077] Appropriate amounts of PRN raw material, PPL raw material, and PRN NB lyophilized powder were subjected to XRD analysis. The operating conditions were set as follows: a Cu target was used as the radiation source, the tube voltage was set to 40 kV, the tube current to 60 mA, the scanning rate was 2° / min, and the scanning angle was 5°–55°. The results are as follows: Figure 7 As shown, PRN active pharmaceutical ingredient exhibits distinct characteristic diffraction peaks between 5° and 25°, indicating that PRN possesses typical crystal structure characteristics; PPL active pharmaceutical ingredient and PRN NB show no diffraction peaks, suggesting that PPL and PRN NB exist in an amorphous state.

[0078] 1.3.4 Determination of PRN NB drug loading:

[0079] 1.3.4.1 Chromatographic conditions: The chromatographic column was a Shim-pack GIS C1000 chromatographic column. 18 (4.6 × 250 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid (30:70); detection wavelength: 249 nm; column temperature: 40 ℃; flow rate: 1.0 min / mL; injection volume: 10 μL.

[0080] 1.3.4.2 Preparation of reference solution: Weigh a certain amount of PRN NB reference standard, accurately weigh it, transfer it to a volumetric flask, add an appropriate amount of methanol, sonicate to dissolve, and then dilute to the mark. Shake well to obtain a 41.0 μg / mL PRN reference solution.

[0081] 1.3.4.3 Preparation of the test solution: Weigh approximately 5 mg of PRN NB lyophilized powder accurately, transfer it to a 100 mL volumetric flask, add an appropriate amount of methanol, dissolve by sonication, and dilute to the mark. Shake well to obtain the test solution.

[0082] 1.3.4.4 Specificity Assessment: PRN reference solution, test solution, and methanol solution were taken separately and analyzed under the chromatographic conditions described in section "1.3.4.1". The results are as follows: Figure 8 As shown, both the PRN reference solution and the test solution showed significant absorption at the same retention time, while the blank solution did not show a significant chromatographic peak. This indicates that other components did not interfere with the determination of PRN, demonstrating the good specificity of this method.

[0083] 1.3.4.5 Linearity Assessment A suitable amount of the PRN control group was precisely pipetted and serially diluted with methanol to prepare a series of mixed reference solutions with different concentration gradients: 4.10, 20.50, 41.00, 61.50, and 82.00 μg / mL. Chromatographic determination was performed according to the conditions described in section "1.3.4.1". The concentration of the reference standard was set as the x-axis, and the peak area as the y-axis. Linear regression analysis was performed, and a standard curve was plotted. The regression equation was y = 47828x + 6798.8, with a correlation coefficient R² of 0.9998. The results indicate that PRN exhibits good linearity within the concentration range of 4.10–82.00 μg / mL.

[0084] 1.3.4.6 Precision Experiment: Accurately pipette a PRN reference solution with a mass concentration of 41.00 μg / mL, and inject it six times under the chromatographic conditions described in section “1.3.4.1”. Record the peak area and calculate the PRN peak area RSD. The results show that the PRN peak area RSD is 0.79%, indicating good instrument precision.

[0085] 1.3.4.7 Repeatability Experiment: Six portions of PRN NB lyophilized powder, approximately 5 mg each, were accurately weighed. The test solution was prepared according to the method described in section "1.3.4.3," and then injected separately under the chromatographic conditions described in section "1.3.4.1." The peak areas were recorded, and the drug content was calculated. The results showed that the PRN content in PRN NB was 45.88%, and the RSD was 1.00%. This indicates that the method has good repeatability.

[0086] 1.3.4.8 Stability Test: The test solution was prepared according to the method described in section 1.3.4.3, and the chromatographic peak area was measured once at 0, 2, 4, 6, 8, 12, and 24 h. The peak area was recorded, and the RSD of the PRN peak area was calculated. The results showed that the RSD of the PRN peak area value of the test solution was 0.14% within 24 h, indicating that the test solution was stable after 24 h.

[0087] 1.3.4.9 Recovery Experiment: Accurately weigh 2.5 mg of each of six known-content PRN NB lyophilized powders into 50 mL volumetric flasks. Add 1 mg of PRN reference standard, dissolve in methanol by sonication, and bring to volume. Filter through a 0.45 μm filter and inject according to the chromatographic conditions in section "1.3.4.1". Calculate the PRN content, recovery rate, and RSD. The results show that the recovery rate of PRN is 97.09–101.30%, and the RSD is 1.61%, indicating that the method has good accuracy.

[0088] 1.3.4.10 Drug Loading Determination: Weigh approximately 5 mg of 6 portions of PRN NB lyophilized powder accurately. Prepare the test solution according to the method in section “1.3.4.3”, and then inject and analyze them according to the chromatographic conditions in section “1.3.4.1”. Substitute the peak area into the regression equation y = 47828x + 6798.8 to calculate the drug loading of PRN. The results show that the drug loading of PRN NB is (46.95 ± 0.52)%.

[0089] 1.3.5 Determination of equilibrium solubility:

[0090] Excess PRN raw material and PRN NB were added to 5 mL of pure water, respectively, and placed in an air bath constant temperature shaker. The mixture was shaken at 37℃ and 100 rpm for 48 h, with three replicates per group. The samples were centrifuged at 13000 rpm for 20 min, and the supernatant was filtered through a 0.45 μm filter membrane and analyzed according to the chromatographic conditions described in section "1.3.4.1". The results are shown in [Figure number missing]. Figure 9 The results showed that the equilibrium solubility of PRN in water was 2568.10 μg / mL, and the equilibrium solubility of PRN NB was 5975.79 μg / mL. After preparing the drug into NB, the solubility increased by 2.33 times.

[0091] 1.4 Stability assessment:

[0092] Three batches of prepared PRN NB samples were placed in a refrigerator at 4 ℃ for 7 days. Samples were taken on days 0, 1, 3, 5, and 7 to determine PS, PDI, Zeta potential, and drug loading. Results are shown below. Figure 10 As shown, the results indicate that PRN NB is stable after being placed at 4 ℃ for one week.

[0093] 1.5 In vitro release study:

[0094] 1.5.1 Chromatographic conditions: The chromatographic column was a Shim-pack GIS C18 (4.6×250mm, 5μm); the mobile phase was methanol-0.1% phosphoric acid (30:70); the detection wavelength was 249 nm; the column temperature was 40 ℃; the flow rate was 1.0 min / mL; and the injection volume was 10 μL.

[0095] 1.5.2 Preparation of reference solution: Accurately weigh PRN reference standard into a volumetric flask, dissolve and dilute with methanol to the mark to prepare a reference solution with a concentration of 41.00 μg / mL.

[0096] 1.5.3 Preparation of simulated gastric juice: Take 2.0 g sodium chloride and 3.2 g pepsin, add 7.0 mL hydrochloric acid and ultrapure water to dissolve to 1000 mL, and the solution is obtained.

[0097] 1.5.4 Preparation of simulated intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate, add 77 mL of 0.2 mol / L sodium hydroxide solution and 500 mL of ultrapure water, then add 10 g of trypsin to dissolve it. Adjust the pH to 6.8 ± 0.1 with 0.2 mol / L sodium hydroxide solution or 0.2 mol / L hydrochloric acid solution, and then dilute with ultrapure water to 1000 mL to obtain the solution.

[0098] 1.5.5 Preparation of test sample: Accurately weigh 5 mg of PRN NB lyophilized powder into a 50 mL volumetric flask, add simulated intestinal fluid, shake on a constant temperature shaker at 37℃ for 1 h, add simulated intestinal fluid to make up the volume, filter through a 0.45 μm filter membrane, and set aside for use.

[0099] 1.5.6 Specificity Assessment: Under the chromatographic conditions described in “1.5.1”, the treated blank simulated intestinal fluid, PRN reference standard, and PRN NB test sample were detected. The chromatograms were recorded, and the results are as follows: Figure 11 As shown, the test solution exhibited a chromatographic peak at the same retention time as the PRN reference standard, while the blank simulated intestinal fluid showed no absorption peak, indicating that the blank simulated intestinal fluid did not interfere with the determination of PRN, demonstrating the good specificity of this method.

[0100] 1.5.7 Linearity Assessment A suitable amount of PRN control group was precisely pipetted and serially diluted with methanol to prepare a series of mixed reference solutions with concentration gradients of 0.41, 2.05, 4.10, 20.50, 41.00, 61.50, and 82.00 μg / mL. The solutions were determined according to the chromatographic conditions described in "1.5.1". Linear regression was performed with the concentration of the reference standard as the abscissa and the peak area as the ordinate, and a standard curve was plotted. The regression equation was y = 47007x + 1910.7, with R² = 1.0000, indicating that PRN exhibits a good linear relationship between 0.41 and 82.00 μg / mL.

[0101] 1.5.8 Precision Test: Accurately pipette a PRN reference solution with a mass concentration of 41.00 μg / mL and inject it six times under the chromatographic conditions described in "1.5.1". Record the peak area and calculate the RSD of the PRN peak area. The results show that the RSD of the PRN peak area is 1.22%, indicating good instrument precision.

[0102] 1.5.9 Stability Test: The test solution was measured at 0, 2, 4, 6, 8, 12, and 24 h, and the peak area was recorded. The RSD of the PRN peak area was calculated. The results showed that the RSD of the PRN peak area was 0.10%, indicating that the test solution was stable within 24 h.

[0103] 1.5.10 Repeatability Test: Accurately weigh 5 mg of PRN NB lyophilized powder into 6 portions to prepare test solutions. Inject each solution according to the chromatographic conditions described in section "1.5.1", record the peak area, and calculate the PRN content and RSD value. The results showed that the PRN content was 451.90 mg / g and the RSD was 1.33%. This indicates that the method has good repeatability.

[0104] 1.5.11 Spiking Recovery Test: Accurately weigh 2.5 mg of each of six known-content PRN NB lyophilized powders into 50 mL volumetric flasks. Add 1.1 mg of PRN reference standard, dissolve in simulated intestinal fluid by sonication, and bring to volume. Filter through a 0.45 μm filter and inject for analysis according to the chromatographic conditions in section "1.5.1". Calculate the PRN content, recovery rate, and RSD. The results show that the recovery rate of PRN is 97.68%–101.68%, and the RSD is 1.55%, indicating that the method has good accuracy.

[0105] 1.5.12 The in vitro release experiment was conducted using Method 3 (small cup method) for the determination of dissolution and release rate as specified in General Chapter 0931, Part IV of the 2020 edition of the Chinese Pharmacopoeia. Simulated gastric and intestinal fluids were used as the dissolution medium, with a rotation speed of 100 rpm and a temperature of 37℃. Appropriate amounts of PRN raw material and PRN NB lyophilized powder (equivalent to 20 mg of PRN) were weighed and placed in a dissolution vessel. Timing was initiated from the moment the powder contacted the liquid surface. 1 mL samples were taken at 5, 10, 20, 30, 45, and 60 min, and simultaneously, the same volume and temperature of simulated intestinal or gastric fluid were added. The samples were filtered through a 0.45 μm microporous membrane and then analyzed under the chromatographic conditions described in section "1.5.1". The cumulative release was calculated, and the results are shown in [Figure 1]. Figure 12 As shown in the figure, PRN NB reached release equilibrium in both simulated gastric and intestinal fluids within approximately 15 minutes. In simulated gastric fluid, PRN API showed a tendency to reach equilibrium after approximately 60 minutes, while in simulated intestinal fluid, PRN API did not reach equilibrium within 60 minutes. In simulated gastric fluid, PRN NB achieved a cumulative release of 85.58% after 60 minutes, while the cumulative dissolution of PRN API was only 33.95%. In simulated intestinal fluid, PRN NB achieved a cumulative dissolution of 85.57% after 60 minutes, while the cumulative dissolution of PRN API was only 30.62%. Compared to PRN API, PRN NB showed a significantly increased cumulative dissolution and a significantly shorter equilibrium time.

[0106] Experimental Example 2: Oral Pharmacokinetic Study in Rats:

[0107] 1.1 Methodology for PRN determination in plasma samples:

[0108] 1.1.1 Preparation of PRN reference standard:

[0109] Accurately weigh 9.60 mg of PRN reference standard, dissolve it in methanol, and dilute to a final volume in a 25 mL volumetric flask to prepare a stock solution of puerarin reference standard with a concentration of 384 µg / mL. Store at 4℃ for later use. Prepare working solutions of puerarin reference standards with concentrations of 15.36 µg / mL, 7.68 µg / mL, 3.84 µg / mL, 1.92 µg / mL, 960 ng / mL, 480 ng / mL, and 240 ng / mL using methanol for the preparation of standard curves for plasma samples.

[0110] 1.1.2 Preparation of internal standard carbamazepine reference standard:

[0111] Accurately weigh 10.74 mg of internal standard (IS) carbamazepine, dissolve it in methanol, and dilute to a final volume of 214.8 µg / mL in a 50 mL volumetric flask. Store at 4°C for later use. When needed, dilute with methanol to obtain a working solution of internal standard with a final concentration of 214.8 ng / mL.

[0112] 1.1.3 Chromatographic and mass spectrometric conditions:

[0113] Chromatographic conditions: The column was a Shimadzu Shim-pack Scepter C18 column (2.1 × 100 mm, 3 μm); mobile phase A was 0.1% formic acid-water, and mobile phase B was methanol, with gradient elution as follows: 0 ~ 1.1 min, 5% B; 1.1 ~ 2.4 min, 5% B ~ 95% B; 2.4 ~ 4.0 min, 95% B; 4.0 ~ 4.5 min, 95% B ~ 5% B; 4.5 ~ 6.0 min, 5% B. Flow rate: 0.3 mL / min; column temperature: 40℃; injection volume: 5 μL.

[0114] Mass spectrometry conditions: ion source was electrospray ionization (ESI); positive ion mode was used; ion source spray voltage was 4.5 kV; ion source temperature was 550 °C; spray gas pressure was 55 psi; auxiliary heating gas pressure was 55 psi; curtain gas pressure was 33 psi; scanning mode was negative ion multiple reaction monitoring (MRM). Specific parameters are shown in Table 1.

[0115] Table 1 Mass Spectrometry Conditions

[0116]

[0117] 1.1.4 Collection of blank plasma samples:

[0118] After rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, samples were collected from the abdominal aorta in anticoagulant tubes containing heparin sodium. After centrifugation at 3000 rpm and 4℃ for 10 min, the supernatant plasma was collected and stored in a -80℃ freezer.

[0119] 1.1.5 Processing of plasma samples:

[0120] The plasma was thawed at room temperature. 100 µL of rat plasma was placed in a 1.5 mL EP tube, 10 µL of internal standard (214 ng / mL) was added, and 290 µL of methanol was added to precipitate the protein. The mixture was vortexed for 3 min, centrifuged at 13000 rpm for 10 min, and 5 µL of the supernatant was injected for LC-MS / MS analysis.

[0121] 1.1.6 Specificity Examination:

[0122] 100 µL of blank rat plasma, blank plasma supplemented with puerarin reference standard and internal standard solution, and plasma samples from rats after gavage administration were collected. The procedure was performed according to section 1.1.5, and PRN and its internal standard were determined under the chromatographic and mass spectrometric conditions described in section 1.1.3. Results are shown below. Figure 13 In plasma samples, the retention time of puerarin was 3.44 min, and the retention time of carbamazepine was 3.92 min. The determination of puerarin and internal standard was not affected by endogenous substances in plasma, and the peak shape was good.

[0123] 1.1.7 Standard Curve Examination:

[0124] 100 µL of blank plasma was precisely pipetted, and 10 µL of PRN working solution and 10 µL of internal standard working solution were added sequentially. 280 µL of methanol was added, and the plasma was processed according to the method described in section 1.1.5. The final concentrations of puerarin in the plasma were 6, 12, 24, 48, 96, 192, and 384 ng / mL. The plasma was analyzed by LC-MS / MS under the chromatographic and mass spectrometric conditions described in section 1.1.3. Linear regression was performed with the ratio of PRN peak area to internal standard peak area (y) as the ordinate and the PRN concentration in the biological sample (x) as the abscissa. The results showed that the standard curve for puerarin was y = 0.0091x + 0.0241, R0. 2 The value was 0.9986, indicating that puerarin showed good linearity in the range of 6 ~ 384 ng / mL.

[0125] 1.1.8 Precision and Accuracy:

[0126] Three control samples of PRN (low, medium, and high concentrations) were prepared according to the procedure described in section 1.1.5. Six samples of each concentration were prepared on the same day and analyzed by LC-MS / MS. The same procedure was followed for three consecutive days, with a standard curve observed daily to determine the concentration of the control samples. The intra-day and inter-day relative standard deviations (RSDs) were used to determine precision, and the ratio of the measured concentration to the true concentration was used to determine accuracy. The precision and accuracy results of PRN are shown in Table 2. In plasma samples, the intra-day precision was ≤ 2.05%, the inter-day precision was ≤ 3.40%, and the accuracy was 99.40% ~ 107.35%, which met the methodological requirements for biological sample analysis.

[0127] Table 2 Precision and accuracy of PRN determination method in quality control samples

[0128]

[0129] 1.1.9 Extraction recovery and matrix effect:

[0130] Accurately pipette 100 µL of blank plasma and prepare PRN quality control plasma samples at low, medium, and high concentrations. Prepare six samples for each concentration and process them according to section 1.1.5. The peak area obtained after LC-MS / MS analysis is denoted as A1. Take blank plasma and, following section 1.1.5, add 10 µL of PRN reference solutions at low, medium, and high concentrations to the biological sample residues, respectively, and inject for analysis. The corresponding peak area is denoted as A2. Under the same analytical conditions, measure the corresponding low, medium, and high concentration reference solutions six times each, and obtain the corresponding peak area as A0. The extraction recovery rate is calculated using the formula A1 / A2, and the matrix effect is calculated using the formula A2 / A0. The results are shown in Table 3. The extraction recovery rate of PRN is 93.22% ~ 105.39%, and the matrix effect of PRN ranges from 93.46% to 98.09%, both of which meet the requirements for biological sample analysis.

[0131] Table 3. Extraction recovery rate and matrix effect of PRN in quality control samples

[0132]

[0133] 1.1.10 Stability:

[0134] Three PRN quality control plasma samples with low, medium, and high concentrations were prepared to investigate the stability of PRN under the following conditions: sample stability was measured after being placed at room temperature for 24 hours; sample stability was measured after being subjected to three freeze-thaw cycles at -80 ℃; and plasma stability was measured after being placed in a -80 ℃ freezer for 30 days. Standard curves were prepared on the day of measurement to calculate the concentration of the quality control samples under the above conditions, and the RSD values ​​were calculated. The results are shown in Table 4. The results show that the RSD of stability under all three conditions is less than 6%, and the results meet the requirements for biological sample analysis.

[0135] Table 4. Stability of PRN in quality control samples

[0136]

[0137] 2.1 Grouping and administration of drugs to rats:

[0138] Twelve male SD rats were randomly divided into a PRN raw material group and a PRN NB group, with six rats in each group. The rats were administered PRN raw material (PRN raw material dissolved in 0.5% sodium carboxymethyl cellulose to prepare a suspension) or PRN NB 200 mg / kg by gavage, respectively.

[0139] 2.2 Collection and processing of plasma samples:

[0140] After 3 days of suitable feeding, all rats were fasted for 12 hours but allowed free access to water before administration. Blood was collected from the retro-orbital vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration. Plasma was separated by centrifugation at 3000 rpm for 10 min at 4°C and stored at -80°C for analysis.

[0141] 2.3 Determination and Results of Plasma Samples:

[0142] Blood samples were processed and PRN concentrations were measured according to the method described in section 1.1.5. The blood drug concentration-time data were processed using the statistical moment model in DAS2.0 software to calculate the main pharmacokinetic parameter AUC of PRN. 0-t C max T max The experimental results are shown in Table 5 and... Figure 14 The results showed that, compared with PRN raw material, PRN NB significantly improved blood drug concentration and bioavailability. P <0.05), as shown in Table 5, compared with PRN raw material, PRN NB has a C max and AUC 0-t The increases were 2.01 times and 1.97 times respectively, indicating that preparing PRN into NB significantly improved the oral absorption of PRN. P<0.05).

[0143] 2.4 Statistical methods:

[0144] Data are expressed as mean ± SD. Statistical analysis was performed using SPSS 20.0 software, and independent samples t-tests were used for comparisons between two groups. P <0.05 indicates a statistically significant difference.

[0145] Table 5 Comparison of pharmacokinetic parameters in rats (M ± SD, n=6)

[0146]

[0147] * P <0.05, ** P <0.01 vs PRN active pharmaceutical ingredient;

[0148] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. Those skilled in the art can make any modifications, alterations, and variations based on the disclosed technical content without departing from the scope of the present invention. These are all equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention still fall within the scope of the present invention.

Claims

1. A homologous polysaccharide-stabilized puerarin nanoribbon, characterized in that, The puerarin nanoribbons were prepared using puerarin and puerarin polysaccharide as raw materials via a media milling method; the specific steps are as follows: (1) Weigh out puerarin raw material and puerarin polysaccharide raw material according to the mass ratio w / w between puerarin raw material and puerarin polysaccharide raw material of 10:2 ~ 10:12, and set aside for later use; (2) Add kudzu polysaccharide to a vial, add water, and after it is completely dissolved, obtain a solution with a kudzu polysaccharide concentration of 0.5~1.5% w / v; (3) Add puerarin to step (2) so that the concentration of puerarin in the solution is 0.5 ~ 1.5% w / v; then add a stir bar and 0.4 ~ 0.6 mm zirconium oxide beads of the same volume as water, place the vial on a magnetic stirrer, and grind at a magnetic stirring speed of 800 ~ 1400 rpm for 2 ~ 12 h, take it out, filter to remove the zirconium oxide beads, and obtain puerarin nanoribbons.

2. The puerarin nanoribbons according to claim 1, characterized in that, The steps are as follows: (1) Weigh 40 mg of puerarin raw material and 40 mg of puerarin polysaccharide for later use; (2) Add kudzu polysaccharide to a 10 mL vial and add 4 mL of water; after it is completely dissolved, a solution with a kudzu polysaccharide concentration of 1% w / v is obtained; (3) Add 40 mg of puerarin to step (2) to make the concentration of puerarin in the solution 1% w / v, then add a stir bar and 4 mL of 0.4 ~ 0.6 mm zirconia beads, place the vial on a magnetic stirrer, set the magnetic stirring speed to 1000 rpm and the grinding time to 4 h, take it out, filter to remove the zirconia beads, and obtain puerarin nanoribbons.

3. The puerarin nanoribbons according to claim 2, characterized in that: The puerarin nanoribbons have a particle size of (174.13 ± 8.11) nm, a polydispersity index of (0.2057 ± 0.0185), and a zeta potential of (-15.29 ± 0.96) mV.

4. The puerarin nanoribbons according to claim 2, characterized in that: The puerarin nanoribbon dispersion is ribbon-shaped, with a width of 50-100 nm and a length of 0.5-2 μm.

5. The puerarin nanoribbons according to claim 2, characterized in that: The drug loading of the puerarin nanoribbons was (46.95 ± 0.52)%.

6. The puerarin nanoribbons according to claim 2, characterized in that: The puerarin nanoribbons showed a cumulative dissolution rate of 85.57% in simulated intestinal fluid over 60 minutes.

7. A method for preparing puerarin nanoribbons stabilized by homologous polysaccharides, comprising the following steps: (1) Weigh out puerarin raw material and puerarin polysaccharide raw material according to the mass ratio w / w between puerarin raw material and puerarin polysaccharide raw material of 10:2 ~ 10:12, and set aside for later use; (2) Add kudzu polysaccharide to a vial, add water, and after it is completely dissolved, obtain a solution with a kudzu polysaccharide concentration of 0.5~1.5% w / v; (3) Add puerarin to step (2) so that the concentration of puerarin in the solution is 0.5 ~ 1.5% w / v; then add a stir bar and 0.4 ~ 0.6 mm zirconium oxide beads of the same volume as water, place the vial on a magnetic stirrer, and grind at a magnetic stirring speed of 800 ~ 1400 rpm for 2 ~ 12 h, take it out, filter to remove the zirconium oxide beads, and obtain puerarin nanoribbons.

8. The preparation method according to claim 7, characterized in that, The steps are as follows: (1) Weigh 40 mg of puerarin raw material and 40 mg of puerarin polysaccharide for later use; (2) Add kudzu polysaccharide to a 10 mL vial and add 4 mL of water; after it is completely dissolved, a solution with a kudzu polysaccharide concentration of 1% w / v is obtained; (3) Add 40 mg of puerarin to step (2) to make the concentration of puerarin in the solution 1% w / v, then add a stir bar and 4 mL of 0.4 ~ 0.6 mm zirconia beads, place the vial on a magnetic stirrer, set the magnetic stirring speed to 1000 rpm and the grinding time to 4 h, take it out, filter to remove the zirconia beads, and obtain puerarin nanoribbons.

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