Preparation method and application of andrographolide prodrug for gastrointestinal positioning drug release

Abstract

The application discloses a preparation method and application of a gastroenteric positioning drug-loaded andrographolide prodrug, and the method comprises the following steps: dispersing the andrographolide prodrug and polyvinylpyrrolidone K30 in purified water, and preparing a crude suspension solution through magnetic stirring, and then preparing a crude crystallization suspension solution through high shear dispersion; preparing nano-crystals of the andrographolide prodrug; dispersing the nano-crystals in soybean oil to prepare an oil phase solution; meanwhile, preparing an aqueous phase solution; slowly adding the oil phase solution into the aqueous phase solution to obtain a drug-containing O / W emulsion; slowly adding the O / W emulsion into a calcium chloride solution with a preset concentration to be cross-linked, solidified and shaped, then washed, coated with Eudragit coating liquid, and finally dried to obtain the gastroenteric positioning drug-loaded andrographolide prodrug. The application can improve the oral absorption efficiency of andrographolide components.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for preparing a gastrointestinal targeted drug delivery prodrug loaded with andrographolide and its application. Background Technology

[0002] Oral administration is one of the clinical routes of drug delivery. However, for drugs classified as BCS Class IV, their oral bioavailability is extremely low due to the dual barriers of poor solubility and low membrane permeability, often necessitating injection in clinical practice. Andrographolide (ADR), a typical BCS Class IV drug, has low absolute oral bioavailability, but its injectable form is strictly limited due to safety concerns. Therefore, effectively improving the oral absorption efficiency of BCS Class IV drugs, especially andrographolide, is a hot and challenging research topic in the field of pharmaceutics.

[0003] In recent years, prodrug technology targeting the oligopeptide transporter (PepT1) has provided a new strategy for improving the oral absorption of low-permeability drugs. PepT1 is highly expressed in intestinal epithelial cells and has a wide substrate range, high transport efficiency, and good species homology. However, existing PepT1-targeted prodrug technologies are mainly applicable to drugs with a certain degree of solubility. For poorly soluble drugs such as andrographolide, the solubilizing effect of the prodrug itself is limited, and insufficient solubility remains one of the key factors restricting oral absorption.

[0004] More importantly, the expression differences of transporters in different regions of the gastrointestinal tract are closely related to the oral absorption efficiency of prodrugs. Previous research by the inventors' team revealed that PepT1 is highly expressed in the upper small intestine (duodenum and jejunum), but rarely in the lower small intestine (ileum) and colon. Conversely, the expression distribution of the efflux transporter P-glycoprotein (P-gp) shows the opposite trend, with low expression in the upper small intestine and gradually increasing towards the ileum. Andrographolide and its prodrug are substrates of P-gp. If the drug is released in the lower small intestine where P-gp is highly expressed, it is easily effluxed into the ileum, significantly reducing absorption efficiency. Furthermore, the inventors' team also found that some andrographolide prodrugs (such as those modified with glycylsarcosine) are easily degraded in the gastric acid environment and under the action of pepsin. If the formulation is released too early in the stomach, the prodrug will be destroyed and cannot effectively reach the absorption site in the small intestine.

[0005] In addition, if the drug is released rapidly in the upper part of the small intestine, it is easy to cause supersaturation of transporters. On the other hand, if the prodrug is exposed to gastrointestinal fluid for too long, the ester bond is easily degraded, which also limits the absorption effect. Summary of the Invention

[0006] This invention provides a method for preparing andrographolide-loaded prodrugs for targeted gastrointestinal drug release and their application, in order to synergistically utilize the microenvironment of the upper small intestine with high PepT1 expression and low P-gp expression to improve the oral absorption efficiency of andrographolide components.

[0007] One aspect of the present invention provides a method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide, comprising: Step S1: Andrographolide prodrug and polyvinylpyrrolidone K30 are dispersed in purified water at a preset mass ratio and mixed evenly by magnetic stirring to obtain a coarse suspension. Then, the coarse suspension is placed in a high-shear disperser and a coarse crystalline suspension solution is obtained by high-shear dispersion. Step S2: The crude crystalline suspension is processed in a high-pressure homogenizer to obtain a nanocrystalline solution of andrographolide prodrug. Then, mannitol is added to the nanocrystalline solution as a freeze-drying protectant. After freeze-drying, andrographolide prodrug nanocrystalline solution is obtained. Step S3: Disperse andrographolide prodrug nanocrystals in soybean oil to obtain an oil phase solution; at the same time, add thiolated chitosan and sodium alginate to purified water at a preset mass ratio, swell, and then heat to dissolve in a constant temperature water bath to obtain an aqueous phase solution. Step S4: Add soybean lecithin as an emulsifier to the aqueous solution and heat to dissolve it. Then, slowly add the oil phase solution to the aqueous solution to obtain a drug-containing O / W emulsion. Step S5: The O / W type emulsion is slowly added dropwise to a calcium chloride solution of a preset concentration, crosslinked and cured for a preset time, then rinsed with ultrapure water, anhydrous ethanol and ultrapure water in sequence, then coated with Eudragit coating solution, and finally dried to obtain a gastrointestinal targeted drug release prodrug loaded with andrographolide.

[0008] The above-mentioned method for preparing andrographolide prodrug with gastrointestinal targeted drug release, wherein in step S1, the mass ratio of andrographolide prodrug to polyvinylpyrrolidone K30 is 1:0.5-1:3.

[0009] The above-mentioned method for preparing a gastrointestinal targeted drug delivery prodrug loaded with andrographolide, wherein in step S1, the crude suspension is placed in a high-shear disperser and sheared at a speed of 14000-17000 rpm for 14-18 min to obtain a crude crystalline suspension solution.

[0010] The above-mentioned method for preparing a gastrointestinal targeted drug delivery prodrug loaded with andrographolide, wherein step S2 specifically includes: The crude crystalline suspension was processed in a high-pressure homogenizer. The high-pressure homogenization process was carried out with the following parameters in sequence: 50 bar for 4 cycles, 200 bar for 4 cycles, 500 bar for 4 cycles, and 1000 bar for 20 cycles to obtain a nanocrystalline solution of andrographolide prodrug. Then, 5% mannitol was added to the nanocrystalline solution as a freeze-drying protectant. After freeze-drying, andrographolide prodrug nanocrystalline solution was obtained.

[0011] The above-mentioned method for preparing a gastrointestinal targeted drug delivery prodrug loaded with andrographolide, wherein step S3 specifically includes: Andrographolide prodrug nanocrystals were dispersed in soybean oil to prepare an oil phase solution; at the same time, thiolated chitosan and sodium alginate were added to purified water at a mass ratio of 2.5:1-25:1, swollen, and then heated in a constant temperature water bath at 50℃ to dissolve, thus obtaining an aqueous phase solution.

[0012] The above-mentioned method for preparing a gastrointestinal targeted drug delivery prodrug loaded with andrographolide, wherein step S4 specifically includes: Soybean lecithin was added to the aqueous solution as an emulsifier and heated to dissolve. The solution was then sheared at 14,000-17,000 rpm for 14-18 min using a high-shear disperser while maintaining the temperature at 50°C. The oil phase solution was then slowly added dropwise to the aqueous phase solution to obtain a drug-containing O / W emulsion.

[0013] The above-mentioned method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide, wherein step S5 specifically includes: The O / W type emulsion was slowly added dropwise to a calcium chloride solution with a concentration of 1-20%, and the cross-linking and curing time was 10-60 min. Then, it was rinsed with ultrapure water, anhydrous ethanol and ultrapure water in sequence. Next, it was coated with Eudragit coating solution and finally dried at room temperature of 25℃ to obtain a gastrointestinal targeted drug release prodrug loaded with andrographolide.

[0014] The above-mentioned method for preparing a gastrointestinal targeted drug delivery prodrug loaded with andrographolide, wherein the Eudragit coating solution is Eudragit L100-55 or Eudragit L30D-55.

[0015] Another aspect of the present invention provides the application of the prodrug prepared by the above method in improving the oral absorption efficiency of andrographolide.

[0016] The present invention has the following beneficial effects: 1. This invention uses PepT1-targeting andrographolide prodrug (ADR-PD) as a raw material to prepare andrographolide prodrug nanocrystals (ADR-PD-NC), which retain the transporter affinity of the prodrug while solubilizing it. The solubility experiment in Example 9 shows that in water at 37°C, the saturated solubility of the andrographolide raw material is only 7.3 μg / mL, and the saturated solubility of its dipeptide prodrug is 61.3 μg / mL, still falling into the category of poorly soluble substances. However, after being prepared into nanocrystals, the saturated solubility of the dipeptide prodrug increased to 803.5 μg / mL, a significant improvement. The in vitro release results in Example 3 further confirm that the cumulative drug release rate of the ADR-PD-NC group in artificial intestinal fluid is significantly higher than that of the free prodrug group. Cellular uptake experiments in Example 6 confirmed that, at the same concentration, the uptake in the ADR-PD-NC group was significantly higher than that in the free prodrug group, indicating that nano-sizing did not weaken the prodrug's PepT1 active recognition and transmembrane transport capabilities. Plasma stability data from Example 10 also showed that the prodrug rapidly degraded after entering the bloodstream (its half-life was significantly shorter than other media), confirming its mechanism of rapid release of the parent drug after recognition by PepT1 to exert its therapeutic effect. These results demonstrate that this invention organically integrates two major strategies: nanocrystal solubilization and active prodrug transport. This not only overcomes the dissolution barrier but also enhances the active transmembrane capacity, providing a dual guarantee for efficient oral absorption.

[0017] 2. This invention uses pH-responsive Eudragit coating material to coat drug-loaded hydrogel microspheres, ensuring the formulation remains intact in the highly acidic environment of the stomach and preventing premature drug leakage. The in vitro release results of Example 3 showed that the ADR-PD-NC@MS group released very little drug in simulated gastric fluid, while the release rate was significantly increased in simulated intestinal fluid, demonstrating the effective barrier effect of the coating layer on gastric fluid. Combined with the stability data from Example 10, the half-life of andrographolide prodrugs differed significantly between rat gastric fluid and simulated gastric fluid. The half-life of some prodrugs was significantly shortened under the action of pepsin; premature release in the stomach would lead to hydrolytic inactivation. This invention, through precise gastric protection and duodenal targeted release design, allows the prodrug to safely pass through the stomach intact, reaching the upper part of the small intestine with high PepT1 expression before release, minimizing inactivation of the prodrug in the cavity before reaching the absorption target.

[0018] 3. This invention embeds nanocrystals within thiolated chitosan-sodium alginate crosslinked hydrogel microspheres, constructing a drug delivery system with both bioadhesive and sustained-release functions. The in vitro intestinal mucosal retention experiment in Example 4 confirmed that the retention rate of ADR-PD-NC@MS modified with thiolated chitosan on the rat intestinal mucosa was significantly higher than that of the unmodified microspheres. This is attributed to the specific adhesion between thiolated chitosan and mucin on the surface of the intestinal mucosa, effectively prolonging the residence time of the formulation in the upper small intestine (the region with high PepT1 expression and low P-gp expression). Simultaneously, the in vitro release curve in Example 3 showed that the microsphere formulation exhibited sustained-release characteristics, avoiding transporter supersaturation and intestinal enzymatic inactivation caused by the instantaneous release of the prodrug. Finally, the in vivo pharmacokinetic results in SD rats in Example 8 showed that, compared to the aqueous solution of andrographolide raw material, free prodrug, and nanocrystals, the ADR-PD-NC@MS of this invention exhibited the best oral absorption effect. The above results fully demonstrate that the present invention significantly improves the oral absorption efficiency of andrographolide prodrug through a multi-level synergistic effect of "solubilization-positioning-adhesion-sustained release". Attached Figure Description

[0019] Figure 1 Scanning electron microscope image of ADR-PD; Figure 2 Transmission electron microscopy image of ADR-PD-NC; Figure 3 Scanning electron microscope image of ADR-PD-NC@MS; Figure 4 The in vitro release curves of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS in artificial gastric fluid are shown. Figure 5 The in vitro release curves of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS in artificial intestinal fluid are shown. Figure 6 In vitro release curves of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS in water; Figure 7 A comparison of the gastric and intestinal retention rates of ADR-PD-NC@MS and ADR-PD-NC@MS-CS in isolated SD rats; Figure 8 The graph shows the changes in retention of ADR-PD-NC@MS-CS in isolated stomach and intestine; Figure 9 Figure showing the results of cytotoxicity studies of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS in MDCK cells; Figure 10Figure showing the results of the study on the uptake levels of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS in MDCK cells; Figure 11 Figure showing the results of tissue distribution studies of ADR-PD-NC and ADR-PD-NC@MS in SD rats; Figure 12 The pharmacokinetic curves of ADR, ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS in SD rats are shown. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more complete description will be given below with reference to various embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] The present invention will be specifically described and verified through different embodiments below. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0023] Example 1 The experimental instruments used in this embodiment are shown in Table 1, and the drugs and reagents are shown in Table 2.

[0024] Table 1

[0025] Table 2

[0026] Example 1 provides a method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide, comprising: In step S1, andrographolide prodrug and polyvinylpyrrolidone K30 are dispersed in purified water at a mass ratio of 1:1 and mixed evenly by magnetic stirring to obtain a crude suspension. Subsequently, the crude suspension is placed in a high-shear disperser and sheared at 16,000 rpm for 15 min to obtain a crude crystalline suspension solution.

[0027] Step S2: The crude crystalline suspension is placed in a high-pressure homogenizer for processing. The high-pressure homogenization process uses the following parameters in sequence: 50 bar for 4 cycles, 200 bar for 4 cycles, 500 bar for 4 cycles, and 1000 bar for 20 cycles to obtain a andrographolide prodrug nanocrystal solution. Further, 5% mannitol is added to the nanocrystal solution as a freeze-drying protectant. After freeze-drying, andrographolide prodrug nanocrystals (ADR-PD-NC) are obtained.

[0028] In step S3, the andrographolide prodrug nanocrystals are dispersed in soybean oil to obtain an oil phase solution. At the same time, thiolated chitosan (CS) and sodium alginate (SA) are added to purified water at a mass ratio of 2.5:1. After swelling, the solution is heated and dissolved in a constant temperature water bath at 50°C to obtain an aqueous phase solution.

[0029] Step S4: Add soybean lecithin as an emulsifier to the aqueous solution and heat to dissolve. Use a high-shear disperser to shear at 16,000 rpm for 15 min while maintaining the temperature at 50°C. Then, slowly add the preheated oil phase dropwise to the aqueous solution and continue shearing to obtain a drug-containing O / W emulsion.

[0030] In step S5, the O / W type emulsion was slowly added dropwise to a 10% calcium chloride solution using a syringe, and cross-linked and cured for 30 min. Then, it was rinsed sequentially with ultrapure water, anhydrous ethanol, and ultrapure water. Next, it was coated with Eudragit L100-55 coating solution with a pH of 5.0 and dried at room temperature of 25°C to finally obtain andrographolide prodrug nanocrystalline hydrogel microspheres (ADR-PD-NC@MS), which are the andrographolide prodrugs loaded with the gastrointestinal targeted drug release.

[0031] In addition, the present invention also provides comparative analysis of different comparative examples 1 to 15.

[0032] Comparative Example 1 The difference between Comparative Example 1 and Example 2 is that the mass ratio of thiolated chitosan (CS) to sodium alginate (SA) in Comparative Example 1 is 10:1.

[0033] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the mass ratio of thiolated chitosan (CS) to sodium alginate (SA) is 25:1.

[0034] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the mass ratio of thiolated chitosan (CS) to sodium alginate (SA) is 30:1.

[0035] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the mass ratio of thiolated chitosan (CS) to sodium alginate (SA) is 1:1.

[0036] Comparative Example 5 The difference between this example and Example 1 is that in Comparative Example 5, the concentration of calcium chloride is 1%.

[0037] Comparative Example 6 The difference between this example and Example 1 is that in Comparative Example 6, the concentration of calcium chloride is 20%.

[0038] Comparative Example 7 The difference between this example and Example 1 is that in Comparative Example 7, the concentration of calcium chloride is 0.5%.

[0039] Comparative Example 8 The difference between this example and Example 1 is that in Comparative Example 8, the concentration of calcium chloride is 23%.

[0040] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is that the crosslinking and curing time in Comparative Example 9 is 10 min.

[0041] Comparative Example 10 The difference between Comparative Example 10 and Example 1 is that the crosslinking and curing time in Comparative Example 10 is 60 min.

[0042] Comparative Example 11 The difference between Comparative Example 11 and Example 1 is that the crosslinking and curing time is 6 min.

[0043] Comparative Example 12 The difference between Comparative Example 12 and Example 1 is that the crosslinking and curing time is 70 min.

[0044] Comparative Example 13 The difference between Comparative Example 13 and Example 1 is that Comparative Example 13 uses Eudragit L30D-55 coating solution with a pH of 5.5 for coating.

[0045] Comparative Example 14 The difference between Comparative Example 14 and Example 1 is that Comparative Example 14 uses Eudragit L100 coating solution with a pH of 6.8 for coating.

[0046] Comparative Example 15 The difference between Comparative Example 1 and Example 1 is that, in Comparative Example 15, Eudragit S100 coating solution with a pH of 7.4 was used for coating.

[0047] The encapsulation efficiency of the andrographolide prodrug nanocrystalline hydrogel microspheres prepared in Example 1 and each comparative example was determined, and the results are shown in Table 3.

[0048] Table 3

[0049] Based on the comparative results of Example 1 and Comparative Examples 1-4, it is evident that an excessively high or low mass ratio of thiolated chitosan to sodium alginate can lead to uneven cross-linking, resulting in incomplete droplet dispersion, affecting appearance and particle size distribution, and causing a decrease in encapsulation efficiency. A higher encapsulation efficiency typically indicates that the drug is uniformly dispersed or bound within the network (e.g., by SA / CS electrostatic adsorption), and release depends on diffusion plus matrix degradation, which is a prerequisite for achieving controlled release and efficient absorption. Therefore, the optimal mass ratio of thiolated chitosan to sodium alginate is 2.5:1 to 25:1.

[0050] Based on the comparison results of Example 1 and Comparative Examples 5-8, it can be seen that a suitable calcium chloride concentration can increase the crosslinking density, densify the network, and improve the encapsulation efficiency. The optimal calcium chloride concentration is 1-20%.

[0051] Based on the comparison results of Example 1 and Comparative Examples 9-12, it can be seen that if the curing time is insufficient, Ca 2+ If the cross-linking is incomplete or the ionic bonds are not fully formed, and the curing time is too long, the network will be over-crosslinked, causing pore shrinkage or even rupture, resulting in a "dehydration shrinkage" effect. The drug will slowly diffuse outward, and the encapsulation efficiency will not be significantly improved. Therefore, the optimal cross-linking curing time is 10-60 minutes.

[0052] Based on the comparison results of Example 1 and Comparative Examples 13-15, it can be seen that if the pH of the coating solution is too high (>6.5), it may cause the precipitation of thiolated chitosan, which will not be able to form a uniform coating layer. Near the isoelectric point (e.g., CS at pH 6.5), the solubility is the lowest and the film formation is poor. Under the optimal charge state (e.g., pH 5.0-5.5), the molecular chains are fully extended to form a dense coating layer, which effectively locks in the drug. Therefore, the preferred Eudragit coating solution is Eudragit L100-55 or Eudragit L30D-55.

[0053] Example 2 The experimental instruments used in this embodiment are shown in Table 4.

[0054] Table 4

[0055] Example 2 describes the morphological examination of andrographolide dipeptide prodrug (ADR-PD), andrographolide prodrug nanocrystals (ADR-PD-NC), andrographolide prodrug nanocrystal hydrogel microspheres (ADR-PD-NC@MS). It should be noted that ADR-PD is specifically a coupled dipeptide prodrug obtained by esterification of the 14-hydroxyl group of andrographolide with the carboxyl group of glycylsarcosine (Gly-Sar).

[0056] The overall morphology of the ADR-PD was observed using SEM. The sample was fixed to the stage using conductive adhesive, sputtered with gold, placed inside the electron microscope sample chamber under vacuum, and the voltage and parameters were adjusted before photographing and observation. Results are referenced below. Figure 1 As shown in the scanning electron microscope image of ADR-PD, it appears as an irregular block with a relatively large particle size, approximately 300-500 μm.

[0057] The overall morphology of ADR-PD-NC was observed using TEM. The sample was ultrasonically dispersed and then dropped onto a copper grid with a carbon film. After drying, staining was possible. The grid was then inserted into the sample holder, and the microscope was evacuated. After adjusting the voltage (typically 80-200 kV), bright-field, dark-field, or high-resolution modes were selected for observation and imaging to analyze its internal ultrastructure, lattice arrangement, and compositional distribution. Results are referenced... Figure 2 As shown in the transmission electron microscope image of ADR-PD-NC, the particle size can reach the nanometer level, exhibiting a near-spherical shape.

[0058] The overall and internal morphology of the ADR-PD-NC@MS sample was observed using SEM. The sample was fixed on the stage with conductive adhesive, sputtered with gold, placed inside the electron microscope sample chamber under vacuum, and the voltage and parameters were adjusted before photographing and observation. Results are referenced below. Figure 3 As shown in the scanning electron microscope image of ADR-PD-NC@MS, the shape is spherical with good roundness and no adhesion.

[0059] Example 3 The experimental instruments used in this embodiment are shown in Table 5, and the drugs and reagents are shown in Table 6.

[0060] Table 5

[0061] Table 6

[0062] Example 3 presents an in vitro release study of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS.

[0063] The in vitro release characteristics of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS in three media—water, simulated gastric fluid, and simulated intestinal fluid—were studied using dialysis. The solubility of the drug in the release medium needed to meet the "leakage condition," so 1% Tween 80 was added to each medium as a surfactant, which can reduce the surface tension of the liquid and increase the saturated solubility of the drug.

[0064] Weigh 0.2 g of sodium chloride, 7 mL of dilute hydrochloric acid (concentration 36.0%), add 3.2 g of pepsin, add 1000 mL of purified water, stir until dissolved, adjust the pH to 1.2 ± 0.5, and prepare artificial stomach simulation solution (SGF); take 6.8 g of potassium dihydrogen phosphate, add water to a final volume of 1 L, add 150 mL of 0.1 mol / L NaOH solution, add 10.0 g of trypsin, stir until dissolved, adjust the pH to 6.8 ± 0.5, and prepare artificial intestine simulation solution (SIF). To ensure full drug release, ADR-PD and ADR-PD-NC were placed in 3500 Da dialysis bags, and ADR-PD-NC@MS was placed in 500 Da dialysis bags. All samples were then placed into centrifuge tubes containing 30 mL of different receiving media, and the ends were tied tightly with string. The temperature was set to 37±2℃, and the tubes were shaken at 100 rpm in a constant-temperature air bath. Samples of 500 μL were taken at 0.5, 1, 2, 4, 6, 8, 12, and 24 h, and an equal volume of preheated (37℃) simulated solution was added. The concentrations were determined by HPLC. All groups were injected in triplicate, and the cumulative drug release rate was calculated.

[0065] Results reference Figures 4 to 6 As shown, the drug release efficiency of the ADR-PD-NC group was significantly higher than that of the ADR-PD group and the ADR-PD-NC@MS group. This indicates that the nanocrystals increased the specific surface area with the release medium, and the nanocrystals could be uniformly dispersed, resulting in a faster drug release rate. The drug release rate of the ADR-PD-NC@MS group was better in the simulated intestinal fluid medium than in the simulated gastric fluid medium. This suggests that using a coating solution may reduce drug release in the stomach and promote drug release under neutral or alkaline intestinal conditions.

[0066] Example 4 The experimental instruments used in this embodiment are shown in Table 7, and the drugs and reagents are shown in Table 8.

[0067] Table 7

[0068] Table 8

[0069] Example 4 investigates the retention of ADR-PD-NC@MS in isolated stomach and intestine.

[0070] To investigate whether the addition of the bioadhesive material thiolated chitosan (CS) can prolong the residence time of drugs at specific sites. Two SD rats were anesthetized by intraperitoneal injection of 4% chloral hydrate. After dissection, the stomach and small intestine were harvested. The stomach was cut open along the greater curvature, and the inner wall was washed with 0.1 mol / L hydrochloric acid. The small intestine was cut open, rinsed with saline, longitudinally cut, and fixed to a glass slide with glue. Following the preparation method provided in Example 1 of this invention, ADR-PD-NC@MS group without CS modification and ADR-PD-NC@MS-CS group with CS modification were prepared by emulsification crosslinking method. A fixed number of microspheres were taken and sprinkled on the gastric mucosa and intestinal mucosa respectively, labeled as N0. They were placed in a sealed container containing 92.5% potassium nitrate saturated solution and the temperature was controlled at room temperature (25±2℃) for about 1 h until swelling. After removal, they were fixed to an inclined plate with double-sided tape, and the gastric tissue was rinsed with 0.1 mol / L hydrochloric acid at a constant flow rate of 12.5 mL / min for 100 mL for a total of 8 min. Collect the flushing fluid, calculate the number of microspheres flushed off (denoted as N), and calculate the retention rate using the following formula. The same procedure applies to the intestinal tract.

[0071] Retention rate = [(N0-N) / N0] × 100%; Please see Figure 7 and Figure 8 The ADR-PD-NC@MS-CS modified with bioadhesive materials showed higher retention in the intestine and lower retention in the stomach compared to the intestine; while the unmodified ADR-PD-NC@MS showed lower retention in isolated stomach and intestine. This indicates that thiolated chitosan can form disulfide bonds with mucins on the surface of mucosal epithelial cells, particularly in regions containing cysteine ​​residues, resulting in specific adhesion and prolonging the close contact time with the mucosal layer.

[0072] Example 5 The experimental instruments used in this embodiment are shown in Table 9, and the drugs and reagents are shown in Table 10.

[0073] Table 9

[0074] Table 10

[0075] Example 5 investigated the cytotoxicity of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS against MDCK.

[0076] Cytotoxicity assays were performed using the CCK8 assay. Log-grown MDCK cells were collected, cell counts were performed, and the cell suspension concentration was adjusted to 8 × 10⁻⁶. 4Cells / mL, take a 96-well plate, add 100 μL of sterile PBS buffer to the outermost ring to prevent liquid evaporation and edge effect, add 100 μL of cell suspension to each of the remaining wells, shake well, and incubate in a cell culture incubator for 24 h to allow the cells to adhere. Prepare ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS solutions, and dilute them sequentially with DMEM to five different concentrations: 50 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, and 1 μg / mL. After 24 hours, remove the 96-well plates and observe under a microscope that the cells have adhered and grown uniformly. Then, administer the drug. Discard the old culture medium, wash three times with PBS buffer, and add 100 μL of each drug solution to create the drug-treated group. Simultaneously, design a control group and a blank group. The control group contains DMEM medium and cells, while the blank group contains only DMEM medium without cells. Incubate in a cell culture incubator for 2 hours and 24 hours, respectively. After incubation, remove the plates, discard the drug solutions, and wash three times with PBS buffer. Under light-protected conditions, add 100 μL of 10% CCK8 solution to each well, shake well, cover the wells with aluminum foil to prevent oxidation, and incubate in a cell culture incubator for 1 hour. h later, the absorbance of each group was measured at a wavelength of 450 nm using a multifunctional microplate reader, and the cell viability was calculated according to the following formula. :

[0077] in, The absorbance of the drug-treated group. The absorbance is for the control group. The absorbance of the blank group is shown.

[0078] Results reference Figure 9 As shown, at 2 h, the cell survival rate of ADR-PD, ADR-PD-NC and ADR-PD-NC@MS groups was above 90% in the concentration range of 1-50 μg / mL, with no obvious toxicity.

[0079] Example 6 The experimental instruments used in this embodiment are shown in Table 11, and the drugs and reagents are shown in Table 12.

[0080] Table 11

[0081] Table 12

[0082] Example 6 investigated the uptake of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS by MDCK cells.

[0083] The uptake of different formulations in MDCK cells was investigated. Log-grown MDCK cells were collected, cell counts were performed, and the cell suspension concentration was adjusted to 4 × 10⁻⁶. 5 Cells were incubated at 37 °C for 24 h in a 5% CO2 incubator, with 0.5 mL of cell suspension added to each well. Cells adhered to the plates, the culture medium was discarded, and the cells were washed three times with PBS buffer. Solutions for ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS were prepared and diluted with DMEM to different concentrations of 20 μg / mL. Cell uptake was assessed after 2 h. Drugs were added in the dark, and after co-incubation in the incubator, the drug solution was discarded. Cells were washed three times with PBS buffer, and 100 μL of 0.25% EDTA trypsin was added to completely detach the cells. Cells were collected into 0.5 mL centrifuge tubes, 100 µL of methanol was added, and the tubes were vortexed for 3 min. The tubes were then centrifuged at 12000 rpm for 5 min. The supernatant was used for HPLC to determine the drug concentration. Protein precipitation was quantified using BCA protein assay. The final uptake result was calculated as the drug content / protein content in the supernatant. Results were referenced. Figure 10 As shown, at the same concentration, the uptake of ADR-PD-NC was higher than that of the ADR-PD group and the ADR-PD-NC@MS group, indicating that changing the particle size of the nanocrystals makes them easier to pass through the cell membrane and accelerates uptake.

[0084] Example 7 The experimental instruments used in this embodiment are shown in Table 13, and the drugs and reagents are shown in Table 14.

[0085] Table 13

[0086] Table 14

[0087] Example 7 uses the DIR probe labeling method to study the in vivo targeting of ADR-PD-NC and ADR-PD-NC@MS. The enrichment degree in the target tissue is determined by quantifying the fluorescence signal.

[0088] DIR probe solution preparation: Accurately weigh 1.0 mg of DIR iodide into a centrifuge tube, add 5 mL of anhydrous ethanol, and sonicate to dissolve. DIR is photosensitive and must be performed under light-protected conditions.

[0089] Preparation of DIR-ADR-PD-NC: The preparation method is the same as described in Example 1 of this invention, except that the purified water in step S1 contains 2 mL of DIR probe solution.

[0090] Preparation of DIR-ADR-PD-NC@MS: Refer to the method described in Example 1 of this invention, except that the purified water in step S1 contains 2 mL of DIR probe solution.

[0091] Four SD rats were acclimatized for one week, with fasting but free access to water for the first 12 hours, and weighing 230±20 g. Two rats were administered DIR-ADR-PD-NC or DIR-ADR-PD-NC@MS formulations by gavage. Two rats in each group were euthanized by dislocation two hours after administration. The small intestine (including duodenum, jejunum, ileum, and colon), heart, liver, spleen, lungs, kidneys, and other major organs were dissected. All dissected tissues were pretreated with physiological saline, fixed with 4% paraformaldehyde solution, and stored at 4°C.

[0092] Take tissue samples from the dissected tissues, cut a partial cross-section, place it face up in an embedding cassette (1 cm in diameter), add an appropriate amount of OCT embedding agent to infiltrate the tissue, and place it in a cryostat for pre-freezing and fixation for about 5 minutes at a temperature of -20°C. Observe that the OCT embedding agent turns white and hardens. Press it out of the embedding cassette and apply 1-2 drops of embedding agent to the sample holder. Place the frozen sample on the sample holder and freeze-fix for 1-2 minutes. Pre-install the anti-roll plate and blade in the appropriate position and angle. Set the section thickness to 10 μm, embed the sample holder in the section position, adjust the angle and distance between the blade and the tissue block, and manually operate the handle of the rotating wheel to section. Attach the sectioned tissue to a glass slide, place it in a box for separate storage, and store it in a -20°C freezer.

[0093] Fluorescence observation and imaging were performed using laser confocal microscopy. Imaging parameters were set as follows: excitation wavelength 748 nm, emission wavelength 780 nm. The slide was inverted under the microscope for observation and imaging. The distribution of fluorescence signals in various tissues, including the small intestine (duodenum, jejunum, ileum, and colon), and major organs such as the heart, liver, spleen, lungs, and kidneys, was analyzed. A blank control group was also included. Tissue bioluminescence was represented by blue fluorescence, and DIR probe-labeled reagents were represented by red fluorescence. ImageJ was used for semi-quantitative analysis of fluorescence intensity.

[0094] Results reference Figure 11As shown, the fluorescence intensity of the DIR-ADR-PD-NC and DIR-ADR-PD-NC@MS groups in the duodenum, jejunum, and ileum was higher than that in the colon, suggesting that the andrographolide prodrug may be released in the proximal intestinal tract. Furthermore, the fluorescence intensity of the DIR-ADR-PD-NC group was weaker than that of the DIR-ADR-PD-NC@MS group, possibly because the nanocrystals exist in crystalline form with a larger specific surface area, resulting in a faster release rate upon contact with body fluids after entering the rat's body. The fluorescence intensity distribution of the DIR-ADR-PD-NC group in various organs was ranked as follows: heart > lung > liver > kidney > spleen. The fluorescence intensity distribution of the DIR-ADR-PD-NC@MS group was ranked as follows: lung > heart > kidney > spleen > liver. This may be because the hydrophobic probe DIR binds more easily to cell membranes or lipid structures, and the heart and lung tissues are rich in cell membranes and lipid components, leading to DIR accumulation in these organs. Additionally, the lungs have a very dense capillary network with high permeability, resulting in a higher concentration of DIR in the lung tissue.

[0095] Example 8 The experimental instruments used in this embodiment are shown in Table 15, and the drugs and reagents are shown in Table 16.

[0096] Table 15

[0097] Table 16

[0098] Example 8: Pharmacokinetic evaluation of ADR-PD, ADR-PD-NC, and ADR-PD-NC@MS.

[0099] In this study, 30 male SD rats weighing 240-260g were randomly divided into 5 groups after one week of acclimatization: ADR-PD injection group, ADR aqueous solution gavage group, ADR-PD gavage group, ADR-PD-NC gavage group, and ADR-PD-NC@MS gavage group. The injection dose was 2 mg / kg, and the gavage dose was 10 mg / kg. Animals were fasted for 12 hours before the experiment, but water was allowed. The SD rats were weighed the following day, and the dosage of the corresponding formulation was calculated. In the gavage groups, blood was collected from the orbital cavity at 5, 15, 30, 45, 60, 120, 240, 360, 480, 720, and 1440 minutes after administration, with each blood volume controlled to be approximately 0.3 mL. Blood was collected using centrifuge tubes containing heparin sodium, and the blood sample was immediately centrifuged at 3500 rpm for 10 min. The supernatant was then transferred to a clean centrifuge tube and stored at 20°C for subsequent analysis of drug concentration in the plasma. Results are as follows. Figure 12As shown, the ADR-PD-NC@MS gavage group had the best effect because it could achieve localized sustained release in the small intestine.

[0100] Example 9 In this embodiment, the saturated solubility of andrographolide (ADR) and andrographolide dipeptide prodrug (ADR-PD) in water was determined using the shake-flask method. The results showed that in water at 37°C, the saturated solubility of andrographolide was 7.3 μg / mL, classifying it as a poorly soluble drug; the saturated solubility of its dipeptide prodrug was 61.3 μg / mL, also falling into the category of poorly soluble drugs. Based on the andrographolide prodrug nanocrystals (ADR-PD-NC) obtained in Example 1, the saturated solubility of its dipeptide prodrug was increased to 803.5 μg / mL; this indicates that nanocrystals can effectively improve the saturated solubility of the prodrug, which is beneficial for its oral absorption.

[0101] Example 10 To determine the stability of the drug under different environments and predict the differences in stability and degradation rate of the prodrug in the gastrointestinal tract and in vivo after oral administration, the stability of andrographolide (ADR), andrographolide dipeptide prodrug (ADR-PD), andrographolide prodrug nanocrystals (ADR-PD-NC) and andrographolide prodrug nanocrystal hydrogel microspheres (ADR-PD-NC@MS) obtained in Example 1 was determined using a timed isothermal co-incubation method. After incubation at 37°C for 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 h, samples were taken and an equal volume of corresponding buffer solution was added. The drug concentration of the samples was determined by HPLC, and the change of prodrug concentration over time was detected to examine the activation ability of the prodrug. The degradation rate constant k was calculated using a first-order kinetic model, and the elimination half-life was calculated using the following formula: t 1 / 2 =0.693 / k; k = (InC0 - InC) / t; Where k is the degradation rate constant, t 1 / 2 The value is the half-life, InC0 is the plasma concentration at the initial time, InC is the plasma concentration at the final time, and t is the time difference between the two times.

[0102] The test results are shown in Table 17: Table 17 Stability of different prodrugs in different media (n=3)

[0103] As shown in Table 17, ADR-PD exhibited good chemical stability in phosphate buffer solutions at pH 1.2 and pH 5.5, indicating that the prodrug is stable in a slightly acidic environment. Its half-lives in artificial intestinal fluid and rat intestinal homogenate were 16.94 ± 0.04 h and 17.01 ± 1.05 h, respectively. This may be due to the slow hydrolysis of chemical bonds in the prodrug structure by specific enzymes such as esterases and peptidases present in the intestine, allowing the prodrug to gradually release the active drug in the intestine, resulting in good overall stability and a long half-life. After preparing the prodrug as nanocrystalline ADR-PD-NC and further loading it onto hydrogel microspheres to obtain ADR-PD-NC@MS, the half-lives of both in the aforementioned acidic buffer solutions and intestinal media were significantly prolonged compared to the free prodrug. Furthermore, the stability enhancement effect of the hydrogel microspheres was more pronounced, indicating that formulation can reduce the direct contact between the prodrug and the hydrolytic environment and enzymes through the blocking and sustained-release effects of the carrier, further delaying degradation. Furthermore, the degradation rate of ADR-PD in rat gastric juice was significantly faster than that in phosphate buffer at the same pH, suggesting that pepsin may hydrolyze the ester bonds in the prodrug structure, making the chemical bonds easier to break in the acidic gastric environment, potentially leading to premature partial release of the active drug or intermediate products. The half-lives of ADR-PD-NC and ADR-PD-NC@MS in both rat gastric juice and artificial gastric juice were significantly longer than those of the free prodrug, effectively resisting degradation by gastric acid and pepsin and improving the stability of the prodrug in the stomach. The ADR active pharmaceutical ingredient had a longer half-life in artificial gastric juice and weakly acidic phosphate buffer, possibly because ADR has low solubility and tends to exist in a solid or aggregated state, thus reducing the contact area with acidic media and lowering the degradation rate. In SD rat plasma, the half-life of ADR-PD was 2.80 ± 0.25 h, significantly shorter than most other media, indicating that the prodrug can be rapidly absorbed into the bloodstream after crossing the small intestinal epithelial cells and can be recognized by the PepT1 transporter, rapidly releasing the parent drug to exert its therapeutic effect. The half-lives of ADR-PD-NC and ADR-PD-NC@MS in rat plasma were 4.32 ± 0.22 h and 5.76 ± 0.29 h, respectively, which were significantly longer than those of the free prodrug. This suggests that formulation can effectively slow down the degradation rate of the prodrug in plasma and prolong its in vivo circulation time.

[0104] In summary, the present invention has the following beneficial effects: 1. This invention uses PepT1-targeting andrographolide prodrug (ADR-PD) as a raw material to prepare andrographolide prodrug nanocrystals (ADR-PD-NC), which retain the transporter affinity of the prodrug while solubilizing it. The solubility experiment in Example 9 shows that in water at 37°C, the saturated solubility of the andrographolide raw material is only 7.3 μg / mL, and the saturated solubility of its dipeptide prodrug is 61.3 μg / mL, still falling into the category of poorly soluble substances. However, after being prepared into nanocrystals, the saturated solubility of the dipeptide prodrug increased to 803.5 μg / mL, a significant improvement. The in vitro release results in Example 3 further confirm that the cumulative drug release rate of the ADR-PD-NC group in artificial intestinal fluid is significantly higher than that of the free prodrug group. Cellular uptake experiments in Example 6 confirmed that, at the same concentration, the uptake in the ADR-PD-NC group was significantly higher than that in the free prodrug group, indicating that nano-sizing did not weaken the prodrug's PepT1 active recognition and transmembrane transport capabilities. Plasma stability data from Example 10 also showed that the prodrug rapidly degraded after entering the bloodstream (its half-life was significantly shorter than other media), confirming its mechanism of rapid release of the parent drug after recognition by PepT1 to exert its therapeutic effect. These results demonstrate that this invention organically integrates two major strategies: nanocrystal solubilization and active prodrug transport. This not only overcomes the dissolution barrier but also enhances the active transmembrane capacity, providing a dual guarantee for efficient oral absorption.

[0105] 2. This invention uses pH-responsive Eudragit coating material to coat drug-loaded hydrogel microspheres, ensuring the formulation remains intact in the highly acidic environment of the stomach and preventing premature drug leakage. The in vitro release results of Example 3 showed that the ADR-PD-NC@MS group released very little drug in simulated gastric fluid, while the release rate was significantly increased in simulated intestinal fluid, demonstrating the effective barrier effect of the coating layer on gastric fluid. Combined with the stability data from Example 10, the half-life of andrographolide prodrugs differed significantly between rat gastric fluid and simulated gastric fluid. The half-life of some prodrugs was significantly shortened under the action of pepsin; premature release in the stomach would lead to hydrolytic inactivation. This invention, through precise gastric protection and duodenal targeted release design, allows the prodrug to safely pass through the stomach intact, reaching the upper part of the small intestine with high PepT1 expression before release, minimizing inactivation of the prodrug in the cavity before reaching the absorption target.

[0106] 3. This invention embeds nanocrystals within thiolated chitosan-sodium alginate crosslinked hydrogel microspheres, constructing a drug delivery system with both bioadhesive and sustained-release functions. The in vitro intestinal mucosal retention experiment in Example 4 confirmed that the retention rate of ADR-PD-NC@MS modified with thiolated chitosan on the rat intestinal mucosa was significantly higher than that of the unmodified microspheres. This is attributed to the specific adhesion between thiolated chitosan and mucin on the surface of the intestinal mucosa, effectively prolonging the residence time of the formulation in the upper small intestine (the region with high PepT1 expression and low P-gp expression). Simultaneously, the in vitro release curve in Example 3 showed that the microsphere formulation exhibited sustained-release characteristics, avoiding transporter supersaturation and intestinal enzymatic inactivation caused by the instantaneous release of the prodrug. Finally, the in vivo pharmacokinetic results in SD rats in Example 8 showed that, compared to the aqueous solution of andrographolide raw material, free prodrug, and nanocrystals, the ADR-PD-NC@MS of this invention exhibited the best oral absorption effect. The above results fully demonstrate that the present invention significantly improves the oral absorption efficiency of andrographolide prodrug through a multi-level synergistic effect of "solubilization-positioning-adhesion-sustained release".

[0107] The above-described embodiments are merely illustrative of several implementation methods of the present invention, and their descriptions are relatively specific and detailed. The proportions of various materials and reaction conditions described above are only exemplary descriptions and should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide, characterized in that, Includes the following steps: Step S1: Andrographolide prodrug and polyvinylpyrrolidone K30 are dispersed in purified water at a preset mass ratio and mixed evenly by magnetic stirring to obtain a coarse suspension. Then, the coarse suspension is placed in a high-shear disperser and a coarse crystalline suspension solution is obtained by high-shear dispersion. Step S2: The crude crystalline suspension is processed in a high-pressure homogenizer to obtain a nanocrystalline solution of andrographolide prodrug. Then, mannitol is added to the nanocrystalline solution as a freeze-drying protectant. After freeze-drying, andrographolide prodrug nanocrystalline solution is obtained. Step S3: Disperse andrographolide prodrug nanocrystals in soybean oil to obtain an oil phase solution; at the same time, add thiolated chitosan and sodium alginate to purified water at a preset mass ratio, swell, and then heat to dissolve in a constant temperature water bath to obtain an aqueous phase solution. Step S4: Add soybean lecithin as an emulsifier to the aqueous solution and heat to dissolve it. Then, slowly add the oil phase solution to the aqueous solution to obtain a drug-containing O / W emulsion. Step S5: The O / W type emulsion is slowly added dropwise to a calcium chloride solution of a preset concentration, crosslinked and cured for a preset time, then rinsed with ultrapure water, anhydrous ethanol and ultrapure water in sequence, then coated with Eudragit coating solution, and finally dried to obtain a gastrointestinal targeted drug release prodrug loaded with andrographolide.

2. The method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide according to claim 1, characterized in that, In step S1, the mass ratio of andrographolide prodrug to polyvinylpyrrolidone K30 is 1:0.5 - 1:

3.

3. The method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide according to claim 1, characterized in that, In step S1, the crude suspension is placed in a high-shear disperser and sheared at 14,000-17,000 rpm for 14-18 minutes to obtain a crude crystalline suspension solution.

4. The method for preparing a prodrug loaded with andrographolide for targeted gastrointestinal drug release according to claim 1, characterized in that, Step S2 specifically includes: The crude crystalline suspension was processed in a high-pressure homogenizer. The high-pressure homogenization process was carried out with the following parameters in sequence: 50 bar for 4 cycles, 200 bar for 4 cycles, 500 bar for 4 cycles, and 1000 bar for 20 cycles to obtain a nanocrystalline solution of andrographolide prodrug. Then, 5% mannitol was added to the nanocrystalline solution as a freeze-drying protectant. After freeze-drying, andrographolide prodrug nanocrystalline solution was obtained.

5. The method for preparing a prodrug loaded with andrographolide for targeted gastrointestinal drug release according to claim 1, characterized in that, Step S3 specifically includes: Andrographolide prodrug nanocrystals were dispersed in soybean oil to prepare an oil phase solution; at the same time, thiolated chitosan and sodium alginate were added to purified water at a mass ratio of 2.5:1-25:1, swollen, and then heated in a constant temperature water bath at 50℃ to dissolve, thus obtaining an aqueous phase solution.

6. The method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide according to claim 1, characterized in that, Step S4 specifically includes: Soybean lecithin was added to the aqueous solution as an emulsifier and heated to dissolve. The solution was then sheared at 14,000-17,000 rpm for 14-18 min using a high-shear disperser while maintaining the temperature at 50°C. The oil phase solution was then slowly added dropwise to the aqueous phase solution to obtain a drug-containing O / W emulsion.

7. The method for preparing a prodrug loaded with andrographolide for targeted gastrointestinal drug release according to claim 1, characterized in that, Step S5 specifically includes: The O / W type emulsion was slowly added dropwise to a calcium chloride solution with a concentration of 1-20%, and the cross-linking and curing time was 10-60 min. Then, it was rinsed with ultrapure water, anhydrous ethanol and ultrapure water in sequence. Next, it was coated with Eudragit coating solution and finally dried at room temperature of 25℃ to obtain a gastrointestinal targeted drug release prodrug loaded with andrographolide.

8. The method for preparing a gastrointestinal targeted drug release prodrug loaded with andrographolide according to claim 1, characterized in that, The Eudragit coating solution is either Eudragit L100-55 or Eudragit L30D-55.

9. The use of the prodrug prepared by the method according to any one of claims 1 to 8 in improving the oral absorption efficiency of andrographolide.

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