A compound sophora flavescens soup nanoparticle delivery system, a preparation method and application thereof

Abstract

The application discloses a compound sophora flavescens soup nanoparticle delivery system and a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method specifically comprises the following steps: (1) preparation of the compound sophora flavescens soup nanoparticle; (2) hyaluronic acid modification; and (3) anion type methacrylic acid copolymer coating. The system can be relatively stable in the stomach and the upper small intestine environment, and can reduce the early release of active ingredients; when reaching the terminal ileum and the colon high-pH environment, the Eudragit S100 outer layer is dissociated in structure, the internal hyaluronic acid modified compound sophora flavescens soup nanoparticle is released, and the colon positioning release of the drug is realized; subsequently, the hyaluronic acid is specifically combined with the cell surface high expression CD44 receptor related to inflammation, the adhesion, retention and cell uptake of the nanoparticle in the colon inflammation area are promoted, and thus the problems of insufficient colon targeting and low lesion enrichment efficiency in the traditional drug delivery mode of the compound sophora flavescens soup are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a compound Kushen decoction nanoparticle delivery system, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease with an incompletely understood etiology. Its pathogenesis involves multiple factors, including intestinal immune imbalance, damage to the intestinal epithelial physical barrier, and intestinal flora dysbiosis. Currently, clinical treatments for UC mainly include aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. While these drugs can alleviate clinical symptoms and control inflammatory responses to some extent, they still have limitations, such as limited clinical remission rates, single-target effects, significant long-term toxic side effects, susceptibility to hormone dependence, and high relapse rates after discontinuation. These limitations make it difficult to meet the clinical needs of UC patients for long-term, safe, and precise treatment. Therefore, developing drugs that combine high efficacy, low toxicity, and targeted delivery has become a crucial issue that urgently needs to be addressed in the treatment of UC.

[0003] Compound traditional Chinese medicine (TCM) formulas possess unique advantages such as multiple components, multiple targets, and minimal toxicity and side effects, showing promising application prospects in the prevention and treatment of diseases like ulcerative colitis (UC). Studies have shown that Compound Sophorae Kushen Decoction (CKD), composed of herbs such as Sophora flavescens, Sanguisorba officinalis, Indigo naturalis, Bletilla striata, Panax notoginseng powder, and Glycyrrhiza uralensis, has good therapeutic effects on UC. However, the pharmacodynamic material basis and mechanism of action of Compound Sophorae Kushen Decoction remain unclear, which to some extent limits its modern development and clinical translation. Furthermore, traditional preparations of Compound Sophorae Kushen Decoction suffer from low bioavailability, insufficient colonic-targeted release capacity, and poor active targeting of inflammation, making it difficult for the drug to achieve effective local accumulation at the lesion site, thus limiting its full therapeutic efficacy. Recent studies have discovered that during the decoction process, various small molecule compounds, polysaccharides, saponins, and organic acids in compound TCM formulas can spontaneously form stable nanostructures through non-covalent interactions such as hydrophobic interactions, π-π stacking, hydrogen bonding, and electrostatic interactions. These structures not only significantly improve drug bioavailability but also achieve "reduced toxicity and enhanced efficacy," making them one of the important material bases for the efficacy of traditional Chinese medicine compound prescriptions. Therefore, isolating and developing nanoparticles with clear pharmacodynamic activity from Compound Kushen Decoction is of great significance for elucidating the material basis of Compound Kushen Decoction in treating ulcerative colitis (UC).

[0004] Therefore, constructing a novel drug delivery system with colon-targeted release and active inflammatory delivery functions is an important strategy to improve the efficacy of Compound Kushen Decoction in treating UC. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a compound Sophora flavescens decoction nanoparticle delivery system, its preparation method and application, so as to overcome the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing a compound Sophora flavescens decoction nanoparticle delivery system specifically includes the following steps: (1) Preparation of compound Kushen decoction nanoparticles Separately weigh out Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, Indigo naturalis, and Panax notoginseng; soak Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, and Indigo naturalis (wrapped in a container) in water, then heat to boiling, decoct, filter, and collect the first decoction; add water again to the dregs, decoct a second time under the same conditions, filter, and collect the second decoction; combine the two decoctions, add Panax notoginseng, and stir continuously under heating conditions to obtain Compound Sophora flavescens Decoction (CKD); centrifuge the Compound Sophora flavescens Decoction and collect the supernatant; centrifuge the supernatant again, discard the supernatant, and collect the precipitate to obtain Compound Sophora flavescens Decoction Nanoparticles (CKD-SANPs), freeze-dry, and store for later use; (2) Hyaluronic acid modification Hyaluronic acid (HA) was dissolved in a first portion of water and stirred to obtain a hyaluronic acid solution. Compound Kushen Decoction nanoparticles were dispersed in a second portion of water to obtain a compound Kushen Decoction nanoparticle dispersion. Under continuous stirring, the compound Kushen Decoction nanoparticle dispersion was added dropwise to the hyaluronic acid solution. After the addition was complete, stirring was continued to obtain a mixture. The mixture was centrifuged, the supernatant was discarded, and the precipitate was collected to obtain hyaluronic acid-modified compound Kushen Decoction nanoparticles (HA-CKD-SANPs). (3) Coating with anionic methacrylic acid copolymer Anionic methacrylic acid copolymer was dissolved in an organic solvent and stirred to form the oil phase. Hyaluronic acid-modified compound Kushen decoction nanoparticles were pre-cooled to form the inner aqueous phase. Polyvinyl alcohol (PVA) aqueous solution was pre-cooled to form the outer aqueous phase. Under ice bath conditions, the inner aqueous phase was added dropwise to the oil phase under ultrasonic probe conditions. After the addition was completed, ultrasonication was continued to form a primary emulsion. The primary emulsion was then added to the outer aqueous phase under stirring to form a secondary emulsion system. Stirring was continued to obtain a suspension. The suspension was centrifuged, the supernatant was discarded and the precipitate was collected. The precipitate was then resuspended in pre-cooled phosphate buffer solution (PBS solution), and centrifuged a second time under the same conditions. The supernatant was discarded and the precipitate was collected to obtain the compound Kushen decoction nanoparticle delivery system (EHK-SANPs).

[0008] In this invention, the compatibility principle of Compound Kushen Decoction is as follows: Kushen is the principal herb, primarily exerting the effects of clearing heat and drying dampness. Sanguisorba officinalis charcoal and Indigo naturalis are the assistant herbs; Sanguisorba officinalis charcoal has the effects of cooling the blood, stopping bleeding, and astringing, while Indigo naturalis has the effects of clearing heat, detoxifying, and cooling the blood. Combined with Kushen, these two herbs enhance the effects of clearing heat, drying dampness, cooling the blood, and detoxifying. Bletilla striata and Panax notoginseng are the adjuvant herbs; Bletilla striata has the effects of astringing, stopping bleeding, reducing swelling, and promoting tissue regeneration, while Panax notoginseng has the effects of resolving blood stasis and stopping bleeding. Combined with Sanguisorba officinalis charcoal, these two herbs combine hemostasis and blood stasis resolution. Licorice is the adjuvant herb, having the effects of clearing heat, detoxifying, relieving spasms, and harmonizing the other herbs. The above-mentioned herbs are used in combination to primarily clear heat, dry dampness, cool the blood, and detoxify, while simultaneously exerting the effects of astringing, stopping bleeding, resolving blood stasis, and promoting tissue regeneration, thus forming a compatibility characteristic that combines clearing heat, drying dampness, cooling the blood, stopping bleeding, and resolving blood stasis and promoting tissue regeneration.

[0009] Furthermore, in step (1) above, the mass ratio of Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, Indigo naturalis and Panax notoginseng is 15:15:10:10:3:3.

[0010] The further beneficial effect of the above-mentioned method is that the preferred raw material composition of the compound Kushen decoction of the present invention is 15g of Kushen (Sophora flavescens), 15g of Sanguisorba officinalis (charred), 10g of Bletilla striata (Bletilla striata), 10g of Glycyrrhiza uralensis (Glycyrrhiza uralensis), 3g of Indigo naturalis (Indigofera tinctoria), and 3g of Panax notoginseng (Panax notoginseng), with a mass ratio of 15:15:10:10:3:3. This formulation uses Kushen and Sanguisorba officinalis (charred) as the main medicinal ingredients, combined with Bletilla striata, Glycyrrhiza uralensis, Indigo naturalis, and Panax notoginseng, so that each medicinal ingredient forms a multi-component synergistic system during the decoction process. It also facilitates the formation of stable compound Kushen decoction nanoparticles by non-covalent interactions such as hydrophobic interactions, hydrogen bonds, electrostatic interactions, and π-π stacking of small molecule compounds, polysaccharides, saponins, and organic acids.

[0011] Furthermore, in step (1) above, the soaking time is 30 min; the decocting time is 30 min; the centrifugation speed is 300×g and the time is 10 min; the centrifugation speed again is 100000×g and the time is 2 h.

[0012] The further beneficial effect of the above-mentioned method is that the compound Kushen decoction of the present invention is composed of Kushen (Sophora flavescens), Sanguisorba officinalis (charred), Bletilla striata (Bletilla striata), Glycyrrhiza uralensis (Glycyrrhiza uralensis), Indigo naturalis (Indigo naturalis), and Panax notoginseng (Panax notoginseng). During the preparation process, the medicinal materials are soaked, decocted, filtered, decocted a second time, and the decoctions are combined to obtain a compound Kushen decoction containing natural nanostructures. Large particle impurities are removed by low-speed centrifugation, and the precipitate is collected by ultracentrifugation to obtain compound Kushen decoction nanoparticles.

[0013] Furthermore, in step (2) above, the ratio of hyaluronic acid, the first part of water, compound sophora flavescens decoction nanoparticles and the second part of water is 100 mg: 30 mL: 50 mg: 5 mL.

[0014] The further beneficial effect of the above-mentioned method lies in the fact that, during the surface modification of hyaluronic acid, it is preferable to use 100 mg of hyaluronic acid and 50 mg of compound Kushen decoction nanoparticles in a mass ratio of 2:1. Specifically, 100 mg of hyaluronic acid is dissolved in 30 mL of pure water, and 50 mg of compound Kushen decoction nanoparticles are dispersed in 5 mL of pure water. Then, the dispersion of compound Kushen decoction nanoparticles is slowly added dropwise to the hyaluronic acid solution while stirring thoroughly. This allows the hyaluronic acid to be adsorbed or bound to the surface of the nanoparticles relatively uniformly, thereby maintaining the stability of the particle dispersion and endowing it with the ability to actively target CD44 receptors at inflammatory sites.

[0015] Furthermore, in step (2) above, the stirring speed is 500 rpm and the time is 12 h; the centrifugation speed is 100000×g and the time is 2 h.

[0016] Furthermore, in step (3) above, the anionic methacrylic acid copolymer is Eudragit S100; the organic solvent is acetone; the mass concentration of the polyvinyl alcohol aqueous solution is 1%; the ratio of Eudragit S100, acetone, hyaluronic acid-modified compound sophora flavescens nanoparticles and polyvinyl alcohol aqueous solution is 200 mg: 20 mL: 10 mL: 200 mL.

[0017] The further beneficial effect of adopting the above is that Eudragit S100 (EUDRAGIT) ® Eudragit S100 is a pH-responsive anionic methacrylic acid copolymer manufactured by Evonik. Its chemical name is methacrylic acid-methyl methacrylate copolymer (1:2), and its CAS number is 25086-15-1. Eudragit S100 is a powdered polymer material composed of methacrylic acid and methyl methacrylate structural units in a molar ratio of approximately 1:2. These structural units can be represented as [-CH2-C(CH3)(COOH)-] and [-CH2-C(CH3)(COOCH3)-], respectively. Eudragit S100 exhibits pH-dependent solubility, with low solubility at lower pH levels and significantly increased solubility above pH 7.0. Therefore, it can be used as a pH-responsive enteric coating material, which is beneficial for protecting the coated nanoparticles and achieving colon-targeted release.

[0018] During the Eudragit S100 enteric coating process, 200 mg of Eudragit S100 was preferably dissolved in 20 mL of acetone as the oil phase (O), with a mass-volume concentration of 10 mg / mL in the oil phase. 10 mL of a hyaluronic acid-modified compound Kushen decoction nanoparticle dispersion was used as the inner aqueous phase (W1), and 200 mL of an aqueous solution containing 1% PVA was used as the outer aqueous phase (W2). The volume ratio of the inner aqueous phase, oil phase, and outer aqueous phase was 1:2:20. This ratio is beneficial for forming relatively stable W1 / O primary emulsion and W1 / O / W2 secondary emulsion, and also facilitates the deposition of Eudragit S100 on the outer layer of HA-CKD-SANPs to form a continuous enteric protective layer. Simultaneously, it reduces particle aggregation and free polymer residue.

[0019] In summary, the present invention preferably uses a mass ratio of Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, Indigo naturalis, and Panax notoginseng of 15:15:10:10:3:3, a mass ratio of hyaluronic acid to compound Sophora flavescens decoction nanoparticles of 2:1, and a volume ratio of inner aqueous phase, oil phase, and outer aqueous phase of 1:2:20. Eudragit S100 at 10 mg / mL is used in the oil phase, and 1% PVA is used in the outer aqueous phase. This raw material ratio can balance the formation of compound Sophora flavescens decoction nanoparticles, the uniformity of hyaluronic acid surface modification, the integrity of the Eudragit S100 coating layer, and the dispersion stability, colon-targeted release performance, and active inflammatory targeting capability of the final nanodelivery system.

[0020] Furthermore, in step (3) above, the hyaluronic acid-modified compound Sophora flavescens nanoparticles are pre-cooled on ice; the polyvinyl alcohol aqueous solution is pre-cooled to 4°C.

[0021] Furthermore, in step (3) above, the power of the continued ultrasound is 100 W, the time is 30-60 s; the stirring time is 4-6 h; the centrifugation temperature is 4℃, the speed is 100000×g, and the time is 2 h.

[0022] The further beneficial effect of the above-mentioned method is that the present invention uses the nanoparticles naturally formed during the decoction of compound Kushen decoction as the pharmacodynamic material basis and carrier core, and constructs hyaluronic acid modified compound Kushen decoction nanoparticles coated with Eudragit S100 through hyaluronic acid surface modification and Eudragit S100 enteric coating, which is a compound Kushen decoction nanoparticle delivery system with colon-localized release and active inflammation targeting functions.

[0023] This invention also claims protection for a compound sophora flavescens decoction nanoparticle delivery system prepared by the above-described method.

[0024] The present invention also claims the use of a compound Kushen decoction nanoparticle delivery system prepared by the above preparation method in the preparation of a drug for treating ulcerative colitis.

[0025] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention addresses the shortcomings of existing ulcerative colitis (UC) treatments, such as single-target action, significant toxic side effects, low bioavailability of Compound Kushen Decoction, insufficient colon-targeted release capacity, and poor accumulation at inflammatory lesions. It provides a Compound Kushen Decoction nanoparticle delivery system that combines colon-targeted release and active targeting of inflammation. This invention isolates naturally formed nanoparticles (CKD-SANPs) from the decoction of Compound Kushen Decoction and uses them as the delivery carrier and pharmacodynamic basis of the compound's active ingredients. To further improve its targeted delivery efficiency at UC lesions, this invention leverages the specific binding between hyaluronic acid and the highly expressed CD44 receptor at the inflammatory site, as well as the responsive release characteristics of Eudragit S100 enteric-coated material under intestinal pH conditions. Through sequential surface modification with HA and stepwise encapsulation with Eudragit S100, a Eudragit S100 / HA-CKD-SANPs (EHK-SANPs) nanodelivery system is constructed. This system can maintain relative stability in the stomach and upper small intestine, reducing premature release of active ingredients. Upon reaching the terminal ileum and the higher pH environment of the colon, the outer layer of Eudragit S100 undergoes structural dissociation, releasing the hyaluronic acid-modified compound Kushen decoction nanoparticles inside, achieving colon-targeted drug release. Subsequently, hyaluronic acid specifically binds to the CD44 receptor highly expressed on the surface of inflammation-related cells, promoting the adhesion, retention, and cellular uptake of nanoparticles in the colonic inflammatory area, thereby solving the problems of insufficient colonic targeting and low lesion enrichment efficiency in the traditional administration of compound Kushen decoction.

[0026] Compared with existing technologies, this invention has advantages such as high preservation of the overall compound composition, clear delivery function, strong targeting, and clear preparation path. Using compound Kushen decoction nanoparticles as the core, this invention can retain the synergistic effects of multiple components and targets of the compound traditional Chinese medicine, and helps to improve the stability and bioavailability of active ingredients. Through the combination of Eudragit S100 enteric coating and hyaluronic acid surface modification, a step-by-step delivery mode of "outer layer colon-targeted release - middle layer inflammation active targeting - core compound synergistic treatment" is formed, allowing the active components to act more concentratedly on the inflammatory lesions of the colon, improving local drug exposure and utilization efficiency, while reducing premature release to non-target sites and potential adverse reactions. Therefore, this delivery system provides a safer, more precise, and more efficient new technical solution for the treatment of ulcerative colitis with compound Kushen decoction. Attached Figure Description

[0027] Figure 1 The particle size distribution and zeta potential analysis of EHK-SANPs in Example 1 are shown below; where A: TEM results of CKD-SANPs; B: TEM results of HA-CKD-SANPs; C: TEM results of EHK-SANPs; D: SEM results of EHK-SANPs; E: DLS results of CKD-SANPs; F: DLS results of HA-CKD-SANPs; G: DLS results of EHK-SANPs; H: Zeta potential map of CKD-SANPs; I: Zeta potential map of HA-CKD-SANPs; J: Zeta potential map of EHK-SANPs. Figure 2 The images show the morphology, elemental analysis, and XPS spectra of the EHK-SANPs in Example 1; where A: HAADF-STEM image of the nanoparticles; B: Overlay distribution map of C, N, and O elements; C: C element distribution map; D: N element distribution map; E: O element distribution map; F: Full XPS spectrum; G: C 1s high-resolution XPS spectrum; H: N 1s high-resolution XPS spectrum; I: O 1s high-resolution XPS spectrum. Figure 3 The Fourier transform infrared spectrum of EHK-SANPs in Example 1; Figure 4 The XRD diffraction pattern and in vitro particle size stability evaluation results of EHK-SANPs in Example 1 are shown below; where A: XRD diffraction pattern of EHK-SANPs; B: Particle size stability diagram of EHK-SANPs. Figure 5 The results show the colonic targeting evaluation of EHK-SANPs in UC mice in Example 1; where A: in vivo and in vitro imaging detection results of mice; B: in vivo imaging fluorescence analysis results of mice 24 h after drug administration; C: fluorescence analysis results of mouse colon 24 h after drug administration. Figure 6 This section describes the alleviating effect of EHK-SANPs on colitis in UC mice in Example 1. A: Photographs of diarrhea and bloody stools in the blank control group; B: Photographs of diarrhea and bloody stools in the model group; C: Photographs of diarrhea and bloody stools in the high-dose group; D: Photographs of diarrhea and bloody stools in the medium-dose group; E: Photographs of diarrhea and bloody stools in the low-dose group; F: Photographs of diarrhea and bloody stools in the positive control group; G: HE staining of the colon in the blank control group; H: HE staining of the colon in the model group; I: HE staining of the colon in the high-dose group; J: HE staining of the colon in the medium-dose group; K: HE staining of the colon in the low-dose group; L: HE staining of the colon in the positive control group; M: Photographs of the large intestine of mice in each group; N: Analysis of the length of the large intestine of mice in each group; O: Western blot image of intestinal barrier-related proteins; P: Mouse colon. ZO-1 mRNA relative expression level detection results; Q: mouse colon Occludin mRNA relative expression level detection results; R: mouse colon Claudin-1 mRNA relative expression level detection results; S: Mouse colon ZO-1 protein expression level detection results; T: Mouse colon Occludin protein expression level detection results; U: Mouse colon Claudin-1 protein expression level detection results; Figure 7 The results of the effects of EHK-SANPs on inflammatory factors of the colon in UC mice in Example 1; where A: mouse colon IL-6 mRNA relative expression level detection results; B: Mouse colon IL-1β mRNA relative expression level detection results; C: mouse colon TNF-α D: Detection results of relative mRNA expression levels; E: Detection results of IL-6 protein expression levels in mouse colon; F: Detection results of IL-1β protein expression levels in mouse colon; Figure 8 This is a flowchart of the construction process of EHK-SANPs in Example 1. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1 The preparation method of the compound Sophora flavescens decoction nanoparticle delivery system specifically includes the following steps: (1) Preparation of compound Kushen decoction nanoparticles Weigh out 15 g of Sophora flavescens, 15 g of Sanguisorba officinalis charcoal, 10 g of Bletilla striata, 10 g of Glycyrrhiza uralensis, 3 g of Indigo naturalis, and 3 g of Panax notoginseng. Soak the Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, and Indigo naturalis (wrapped in a container) in cold distilled water for 30 min. Then heat to boiling over high heat, then simmer over low heat for 30 min. Filter and collect the first decoction. Add distilled water to the dregs again and decoct under the same conditions. Filter and collect the second decoction. Combine the two decoctions and add Panax notoginseng. Stir continuously under heating to ensure that the Panax notoginseng powder is fully mixed and evenly dispersed with the decoction, obtaining Compound Sophora flavescens Decoction (CKD). Transfer it to a centrifuge tube and centrifuge at 300×g for 10 min to remove dregs, suspended impurities, and other large particles. Collect the supernatant. Then ultracentrifuge the supernatant at 100000×g for 2 seconds. h, discard the supernatant and collect the precipitate to obtain compound sophora flavescens decoction nanoparticles (CKD-SANPs), freeze-dry and store for later use; (2) Hyaluronic acid modification 100 mg of hyaluronic acid was dissolved in 30 mL of pure water and thoroughly mixed under mechanical stirring to obtain a homogeneous hyaluronic acid solution. Separately, 50 mg of Compound Kushen Decoction nanoparticles were dispersed in 5 mL of pure water to obtain a Compound Kushen Decoction nanoparticle dispersion. The Compound Kushen Decoction nanoparticle dispersion was slowly added dropwise to the hyaluronic acid solution while maintaining continuous stirring. After the addition was complete, stirring was continued for 12 h under mechanical stirring at 500 rpm to allow hyaluronic acid to bind to the surface of the Compound Kushen Decoction nanoparticles, resulting in a mixed solution. Subsequently, the mixture was centrifuged at 100,000 × g for 2 h, and the supernatant was discarded to obtain hyaluronic acid-modified Compound Kushen Decoction nanoparticles (HA-CKD-SANPs). (3) Eudragit S100 coating 200 mg of Eudragit S100 was dissolved in 20 mL of acetone and magnetically stirred until completely clear and transparent, serving as the oil phase (O). 10 mL of hyaluronic acid-modified compound Kushen decoction nanoparticle dispersion (HA-CKD-SANPs) was pre-cooled on ice, serving as the inner aqueous phase (W1). Separately, 200 mL of an aqueous solution containing 1% PVA was prepared and pre-cooled to 4°C, serving as the outer aqueous phase (W2). Under ice bath conditions, the inner aqueous phase (W1) was slowly added dropwise to the oil phase (O) under ultrasonic probe conditions. After the addition was complete, ultrasonication was continued at a medium power of 100 W for 60 s to form a uniform milky white W1 / O pre-emulsion. Subsequently, the obtained W1 / O pre-emulsion was rapidly added to the pre-cooled outer aqueous phase (W2) under high-speed magnetic stirring to form a W1 / O / W2 complex emulsion system. Medium-speed magnetic stirring was continued for 6 h to allow the acetone to fully evaporate. As the solvent evaporated, the system gradually changed from turbid to translucent or opalescent, indicating that Eudragit S100... The S100 polymer shell is formed, resulting in a suspension. The suspension is transferred to an ultracentrifuge tube and centrifuged at 4°C and 100,000 × g for 2 hours. The supernatant is discarded, and the precipitate is collected. The precipitate is then gently resuspended in pre-cooled PBS and centrifuged again under the same conditions for 2 hours to remove free PVA and uncoated polymer. The supernatant is discarded, and the precipitate is collected. The final precipitate is the Eudragit S100-coated hyaluronic acid-modified compound Kushen decoction nanoparticles, also known as the compound Kushen decoction nanoparticle delivery system (EHK-SANPs).

[0030] Performance testing The following performance tests were conducted on the compound Kushen decoction nanoparticles (CKD-SANPs), hyaluronic acid-modified compound Kushen decoction nanoparticles (HA-CKD-SANPs), and compound Kushen decoction nanoparticle delivery system (EHK-SANPs) prepared in Example 1.

[0031] 1. Construction and Preliminary Characterization of EHK-SANPs This invention uses Compound Kushen Decoction as a drug model. Based on the characteristics of the active ingredients in traditional Chinese medicine, Compound Kushen Decoction nanoparticles (CKD-SANPs) were prepared through decoction and centrifugation. Subsequently, hyaluronic acid (HA) surface modification and Eudragit S100 enteric coating were performed sequentially to successfully construct EHK-SANPs with targeted delivery function for colitis. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) observations showed that EHK-SANPs were regular spherical with uniform morphology. Figure 1(AD). Dynamic light scattering (DLS) analysis showed that the hydrodynamic particle sizes of CKD-SANPs, HA-CKD-SANPs, and EHK-SANPs were approximately 103 nm, 124 nm, and 215 nm, respectively, exhibiting a gradually increasing trend, consistent with the characteristics of a multi-level modified structure. Figure 1 (EG). Zeta potential measurements showed that with HA modification and Eudragit S100 coating, the particle surface potential gradually decreased from -13 mV to -25 mV and -34 mV ( Figure 1 (HJ), confirming the successful assembly of each functional layer. Eudragit S100 responsively dissolves in the weakly alkaline environment of the intestine, endowing the nanoparticles with colon-targeted release properties; HA modification enables the nanoparticles to actively target inflamed tissues by binding to the CD44 receptor, which is highly expressed at inflammatory sites. These results demonstrate that EHK-SANPs were successfully constructed, possessing favorable structural characteristics and targeted delivery potential.

[0032] 2. Morphological and elemental distribution analysis of EHK-SANPs Based on the successful construction of EHK-SANPs, their morphology and elemental composition were further characterized using high-resolution transmission electron microscopy combined with energy dispersive spectroscopy (HRTEM-EDS). The results showed that the nanoparticles were regularly spherical, and characteristic elements such as C, N, and O were detected on their surface, consistent with the composition of the raw materials. Figure 2 (Central AE). X-ray photoelectron spectroscopy (XPS) full-spectrum scanning further confirmed that the particle surface mainly contains C, N, and O elements (C, N, O). Figure 2 In the high-resolution C 1s spectrum, the characteristic peaks at 284.8 eV, 286.3 eV, and 288.8 eV correspond to CC / CH, CO / CN, and OC=O bonds, respectively. The CO / CN signal intensity is relatively high, mainly originating from the sugar ring structure of HA, the ester / ether bond of Eudragit S100, and the hydroxyl and glycosidic bonds in the traditional Chinese medicine components. The OC=O signal originates from the ester group of Eudragit S100, the carboxyl group of HA, and the organic acid components of the traditional Chinese medicine (…). Figure 2 The pyridine nitrogen (~398.7 eV) and pyrrole nitrogen (~399.5 eV) signals detected in the N 1s spectrum directly indicate the exposure of traditional Chinese medicine components (such as sophora flavescens alkaloids, indigo / indirubin in indigo) on the particle surface. Figure 2 The C=O (532.0 eV) and CO (533.2 eV) peaks in the O 1s spectrum corroborate the C 1s results, further supporting the enrichment of oxygen-containing functional groups on the surface. Figure 2(I). The above results indicate that EHK-SANPs have a clear core-shell structure, complete functional layer assembly, and effective exposure of the core components of traditional Chinese medicine to the surface proximal layer, laying a structural foundation for subsequent oral colon-targeted delivery and anti-inflammatory activity studies.

[0033] 3. Mechanism Exploration of EHK-SANPs To investigate the chemical composition and mechanism of nanoparticles, FTIR was used for analysis, with the spectral density in the 3000-3500 cm⁻¹ range being analyzed. -1 A broad, strong absorption band is observed at this location (center approximately 3400 cm). -1 The fingerprints, primarily attributed to OH stretching vibrations (with a small amount of NH superposition), confirm that the particle surface is rich in hydroxyl groups. These hydroxyl groups originate from flavonoids and saponins in Sophora flavescens and licorice, as well as polysaccharides from Bletilla striata and saponins from Panax notoginseng. They construct a hydrophilic shell through hydrogen bonding interactions, while the NH signal mainly comes from the secondary amine groups of Sophora flavescens alkaloids and the secondary amide groups of indigo in Indigofera tinctoria. The fingerprint region is 1000-1500 cm². -1 It exhibits complex overlapping peaks, with the ~1600 cm⁻¹ peak being particularly prominent. -1 and ~1500 cm -1 The absorption is attributed to the C=C skeleton vibration of the aromatic ring, suggesting the presence of conjugated aromatic structures such as indigo / indirubin and flavonoids in indigo, 1200-1000 cm⁻¹ -1 Strong absorption within this range corresponds to CO and COC stretching vibrations, primarily originating from the glycosidic bonds and hydroxyl groups of polysaccharides (such as Bletilla striata polysaccharides) and saponins (such as glycyrrhizic acid and Panax notoginseng saponins). Figure 3 Based on comprehensive FTIR analysis, it can be inferred that CKD-SANPs follow a "hydrophobic-driven-hydrogen bond stabilization" mechanism: planar large π-conjugated molecules such as indigo form a hydrophobic core through π-π stacking, while hydroxyl-rich saponins and polysaccharides build a hydrophilic shell around them through a hydrogen bond network, thereby forming a stable nanomicelle structure.

[0034] 4. Evaluation of the crystal structure and colloidal stability of EHK-SANPs To further clarify the internal structure of the nanoparticles, X-ray diffraction (XRD) was used to analyze their crystalline state. The results showed that the spectra exhibited a broad, diffuse diffraction peak in the low-angle region (2θ ≈ 5°-30°), displaying typical amorphous characteristics, indicating a long-range disordered structure within the particles. This structure is beneficial for improving drug dispersibility and dissolution behavior. Figure 4 (A). Regarding stability, dynamic light scattering (DLS) technology was used to monitor particle size for 7 days in both phosphate-buffered saline (PBS) and serum. The results showed no significant fluctuation in particle size, indicating that EHK-SANPs possess good colloidal stability and adaptability to physiological environments. Figure 4 (B). The above results further support its feasibility as an oral colon-targeted delivery system and provide important evidence for subsequent in vivo studies.

[0035] 5. In vivo distribution study of EHK-SANPs in UC mice To further observe whether EHK-SANPs achieve targeted accumulation in UC mice, 18 6-8 week old SPF-grade male BALB / c mice were randomly divided into three groups of 6 mice each: a free indocyanine green group (indocyanine green was used as a near-infrared fluorescent tracer to track the distribution and colonic accumulation of each preparation in mice), an indocyanine green-labeled CKD-SANPs group, and an indocyanine green-labeled EHK-SANPs group. After 3 days of acclimatization, each group was given free access to 5% sodium dextran sulfate (DSS) solution for 5 days to establish an ulcerative colitis model. After modeling, the above preparations were administered by gavage to each group at an equivalent dose of 1 mg / kg of indocyanine green. Based on the indocyanine green loading (2.97% for indocyanine green-labeled CKD-SANPs and 1.85% for indocyanine green-labeled EHK-SANPs), the dosages for the three groups were 1 mg / kg, 33.67 mg / kg, and 54.05 mg / kg, respectively. In vivo imaging was performed at 0, 1, 6, 12, and 24 hours after administration. The results showed that 24 hours after administration, the fluorescence intensity in the indocyanine green-labeled EHK-SANPs group was significantly higher than that in the indocyanine green-labeled CKD-SANPs group (P < 0.05). Figure 5 (AB); 24 h after drug administration, mice in each group were dissected and their hearts, livers, spleens, lungs, kidneys, and large intestines were collected. The distribution of fluorescence in each organ was observed in vitro. The results showed that compared with the indocyanine green-labeled CKD-SANPs group, the fluorescence intensity in the large intestine of mice in the indocyanine green-labeled EHK-SANPs group was significantly higher than that in the indocyanine green-labeled CKD-SANPs group (P < 0.05), while the fluorescence intensity in the heart, liver, spleen, lungs, and kidneys was weaker. Figure 5 (A and C). This indicates that EHK-SANPs have good colon targeting in UC mice compared to CKD-SANPs.

[0036] 6. The alleviating effect of EHK-SANPs on colitis in UC mice Forty-eight SPF-grade BALB / c mice were randomly divided into four groups: a blank control group, an UC model group, a high-dose EHK-SANPs group (10 mg / kg), a medium-dose EHK-SANPs group (5 mg / kg), a low-dose EHK-SANPs group (2.5 mg / kg), and a mesalazine positive control group (100 mg / kg), with eight mice in each group. After acclimatization for three days, all groups except the blank control group were given free access to distilled water containing 5% sodium dextran sulfate (DSS) for five consecutive days to establish the UC mouse model. Drug treatment was administered simultaneously from the start of model establishment. The high, medium, and low-dose EHK-SANPs groups were administered the corresponding doses of EHK-SANPs by gavage, the mesalazine positive control group was administered 100 mg / kg mesalazine by gavage, and the blank control group and the UC model group were administered an equal volume of physiological saline by gavage. All groups were treated for five consecutive days.

[0037] During the treatment period, the fecal characteristics and hematochezia of mice in each group were observed. The results showed that the model group mice had obvious and severe hematochezia, the mesalazine positive control group only had mild hematochezia, and the low-dose EHK-SANPs group mainly showed diarrhea. The blank control group and the medium and high-dose EHK-SANPs groups had basically normal fecal characteristics and no obvious hematochezia. This indicates that EHK-SANPs can improve colitis-related symptoms such as diarrhea and hematochezia induced by DSS, and the improvement effect is more obvious in the medium and high-dose groups. Figure 6 (AF).

[0038] Further HE staining of colon tissues from each group of mice revealed that, compared with the blank control group, the colonic mucosal structure of the model group mice was significantly damaged, characterized by disordered crypt structure, reduced goblet cells, and significant inflammatory cell infiltration. After EHK-SANPs intervention, the aforementioned histopathological changes in each dose group were improved to varying degrees, with the medium and high dose groups showing more significant recovery of colonic tissue structure and a marked reduction in mucosal damage and inflammatory cell infiltration. The colonic tissue damage in the mesalazine-positive control group also improved, but local mucosal damage and inflammatory cell infiltration were still observed. Figure 6 (Chinese GL).

[0039] The length of the large intestine in each group of mice was measured. The results showed that, compared with the blank control group, the large intestine of the model group mice was significantly shortened. After intervention with EHK-SANPs or mesalazine, the shortening of the large intestine was improved to varying degrees. Among them, the recovery of large intestine length was more obvious in the medium and high dose groups of EHK-SANPs, and in terms of overall recovery effect, EHK-SANPs was better than the mesalazine positive control group. Figure 6 (MN).

[0040] To further evaluate the effects of EHK-SANPs on intestinal mucosal barrier function, the expression levels of tight junction-related proteins ZO-1, Occludin, and Claudin-1 in colonic tissue were detected, and the protein immunoblot bands were analyzed (…). Figure 6 Analysis of the results showed that, compared with the blank control group, the mRNA and protein expression levels of ZO-1, Occludin, and Claudin-1 in the model group were significantly reduced (P < 0.05); after intervention with EHK-SANPs or mesalazine, the mRNA and protein expression levels of the above tight junction-related proteins were restored to varying degrees, with the restoration effect of each dose group of EHK-SANPs being more significant overall. Figure 6 (Middle PU). This indicates that EHK-SANPs can promote the recovery of expression of intestinal mucosal barrier-related proteins induced by DSS in UC mice, which is beneficial to improving the damaged intestinal mucosal barrier function.

[0041] Further examination of the expression levels of inflammatory factors IL-6, IL-1β, and TNF-α in colon tissue revealed that, compared with the blank control group, the mRNA and protein expression levels of IL-6, IL-1β, and TNF-α in the model group were significantly increased (P < 0.05). After intervention with EHK-SANPs or mesalazine, the mRNA and protein expression levels of the above inflammatory factors were significantly reduced, and the EHK-SANPs dosage groups showed a more significant downregulation effect than mesalazine. Figure 7 (AF).

[0042] In summary, EHK-SANPs effectively ameliorated DSS-induced diarrhea, bloody stools, and colonic tissue damage in ulcerative colitis mice, alleviated colonic shortening, and promoted the recovery of expression of intestinal mucosal barrier-related proteins such as ZO-1, Occludin, and Claudin-1. Simultaneously, they reduced the expression levels of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, thereby promoting intestinal mucosal barrier repair and inhibiting colonic inflammation. These results indicate that EHK-SANPs have a good alleviating effect on DSS-induced ulcerative colitis, and under the conditions of this experiment, its overall improvement effect is superior to that of mesalazine.

[0043] The key design feature of this invention lies in the fact that multiple active ingredients in Compound Kushen Decoction spontaneously form nanoscale aggregate structures during the decoction process through non-covalent interactions such as hydrophobic interactions, hydrogen bonds, electrostatic interactions, and π-π stacking. These natural nanoparticles not only retain the overall pharmacodynamic characteristics of Compound Kushen Decoction with multiple components and multiple targets, but also serve as endogenous delivery carriers for active components, improving the stability, bioavailability, and local accumulation capacity of the pharmacological substances in the colon. Based on this, the present invention uses hyaluronic acid to modify the surface of CKD-SANPs. Utilizing the specific binding between hyaluronic acid and the highly expressed CD44 receptor at the ulcerative colitis lesion site and inflammation-related cells, it promotes the enrichment and cellular uptake of nanoparticles in colonic inflammation tissue, achieving active targeted delivery to the inflammatory site. Simultaneously, the pH-responsive enteric-coated material Eudragit S100 is used to coat the HA-CKD-SANPs, ensuring the delivery system remains relatively stable in the acidic or weakly acidic environment of the stomach and upper small intestine, reducing premature release and degradation of active components. Upon reaching the terminal ileum and the higher pH environment of the colon, dissolution or structural dissociation occur, releasing the internal HA-CKD-SANPs, thereby achieving colonic-targeted release of the active components of the compound Kushen decoction. Figure 8 ).

[0044] Through the above structural design, this invention forms a delivery mode of "colonic localization and release, active targeting of inflammation". This delivery system can improve the local exposure level, lesion enrichment degree and cellular uptake efficiency of the active substances of Compound Kushen Decoction at the site of colonic inflammation, enhance its therapeutic effect on ulcerative colitis, and at the same time reduce the ineffective release of active ingredients in non-lesion sites and potential adverse reactions.

[0045] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a compound Sophora flavescens decoction nanoparticle delivery system, characterized in that, Specifically, the following steps are included: (1) Preparation of compound Kushen decoction nanoparticles Weigh out Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, Indigo naturalis, and Panax notoginseng separately; soak Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, and Indigo naturalis (wrapped in a decocting bag) in water, then heat to boiling, decoct, filter and collect the first decoction; add water to the dregs again, decoct a second time under the same conditions, filter and collect the second decoction; combine the two decoctions and add Panax notoginseng, stirring continuously under heating conditions to obtain compound Sophora flavescens decoction; The compound Kushen decoction was centrifuged and the supernatant was collected. The supernatant was centrifuged again, the supernatant was discarded and the precipitate was collected to obtain compound Kushen decoction nanoparticles, which were freeze-dried and stored for later use. (2) Hyaluronic acid modification Hyaluronic acid was dissolved in the first part of water and stirred to obtain a hyaluronic acid solution; Compound Kushen Decoction nanoparticles were dispersed in the second part of water to obtain a Compound Kushen Decoction nanoparticle dispersion. Under continuous stirring, the dispersion of compound Kushen decoction nanoparticles was added dropwise to the hyaluronic acid solution. After the addition was completed, stirring was continued to obtain a mixture. The mixture was centrifuged, the supernatant was discarded and the precipitate was collected to obtain hyaluronic acid modified compound Kushen decoction nanoparticles. (3) Coating with anionic methacrylic acid copolymer Anionic methacrylic acid copolymer was dissolved in an organic solvent and stirred to form the oil phase. Hyaluronic acid-modified compound Sophora flavescens nanoparticles were pre-cooled to form the inner aqueous phase. Polyvinyl alcohol aqueous solution was pre-cooled to form the outer aqueous phase. Under ice bath conditions, the inner aqueous phase was added dropwise to the oil phase under ultrasonic conditions with a probe. After the addition was completed, ultrasonication was continued to form the primary emulsion. Subsequently, the primary emulsion was added to the outer aqueous phase under stirring to form a complex emulsion system. Stirring was continued to obtain a suspension. Centrifuge the suspension, discard the supernatant and collect the precipitate; then resuspend the precipitate in pre-cooled phosphate buffer solution, centrifuge a second time under the same conditions, discard the supernatant and collect the precipitate to obtain the compound Sophora flavescens decoction nanoparticle delivery system.

2. The preparation method of the compound Kushen decoction nanoparticle delivery system according to claim 1, characterized in that, In step (1), the mass ratio of Sophora flavescens, Sanguisorba officinalis charcoal, Bletilla striata, Glycyrrhiza uralensis, Indigo naturalis and Panax notoginseng is 15:15:10:10:3:

3.

3. The preparation method of the compound Kushen decoction nanoparticle delivery system according to claim 1, characterized in that, In step (1), the soaking time is 30 min; the decocting time is 30 min; the centrifugation speed is 300×g and the time is 10 min; the second centrifugation speed is 100000×g and the time is 2 h.

4. The preparation method of the compound Kushen decoction nanoparticle delivery system according to claim 1, characterized in that, In step (2), the ratio of hyaluronic acid, the first part of water, compound sophora flavescens nanoparticles and the second part of water is 100 mg: 30 mL: 50 mg: 5 mL.

5. The preparation method of the compound Kushen decoction nanoparticle delivery system according to claim 1, characterized in that, In step (2), the stirring speed is 500 rpm and the time is 12 h; the centrifugation speed is 100000×g and the time is 2 h.

6. The preparation method of the compound Kushen decoction nanoparticle delivery system according to claim 1, characterized in that, In step (3), the anionic methacrylic acid copolymer is Eudragit S100; the organic solvent is acetone; the mass concentration of the polyvinyl alcohol aqueous solution is 1%; the ratio of Eudragit S100, acetone, hyaluronic acid-modified compound sophora flavescens nanoparticles and polyvinyl alcohol aqueous solution is 200 mg: 20 mL: 10 mL: 200 mL.

7. The preparation method of the compound Kushen decoction nanoparticle delivery system according to claim 1, characterized in that, In step (3), the hyaluronic acid-modified compound Sophora flavescens nanoparticles are pre-cooled on ice; the polyvinyl alcohol aqueous solution is pre-cooled to 4°C.

8. The preparation method of the compound Kushen decoction nanoparticle delivery system according to claim 1, characterized in that, In step (3), the power of the continued ultrasound is 100 W and the time is 30-60 s; the time of continued stirring is 4-6 h; the temperature of the centrifugation is 4℃, the speed is 100000×g, and the time is 2 h.

9. A compound Sophora flavescens decoction nanoparticle delivery system prepared by the preparation method according to any one of claims 1-8.

10. The use of a compound Kushen decoction nanoparticle delivery system prepared by any one of claims 1-8 in the preparation of a medicament for treating ulcerative colitis.

Images