8-O-acetyl harpagide nano composition and preparation method thereof

Abstract

The invention belongs to the field of drug delivery and oral preparations, and provides an 8-O-acetyl harpagide nano-composition and a preparation method thereof.The 8-O-acetyl harpagide-soybean lecithin compound is used as a nuclear phase, lactobionic acid and thioglycolic acid dual-modified chitosan and sodium tripolyphosphate are subjected to ionic crosslinking to form a shell layer, and the 8-O-acetyl harpagide nano-composition is prepared. Compared with the prior art, the freeze-dried powder has the advantages of high loading capacity, high solid content, low viscosity and wide processing window, small particle size, low polydispersity index and stable nano dispersion are obtained, ethanol residue and water content of the freeze-dried powder are reduced, freeze-dried storage stability, redissolution redispersibility and oral release and absorption performance are improved, and the freeze-dried powder is suitable for large-scale production. The method is suitable for the development of oral preparations for preventing the acetaminophen-induced liver injury.

Description

Technical Field

[0001] This invention relates to the field of drug delivery and oral nanoformulation, specifically to an 8-O-acetylharbazoside nanocomposition and its preparation method. Background Technology

[0002] Acetaminophen is a commonly used antipyretic and analgesic, but it can cause liver damage when the recommended dose is exceeded. Therefore, developing 8-O-acetylharbazoside formulations suitable for oral administration with stable dispersion and good release and absorption characteristics is of practical significance. When using 8-O-acetylharbazoside in related drug development, it is necessary to consider not only the composite stability between 8-O-acetylharbazoside and soybean lecithin, but also the loading, solid content, low viscosity, and wide processing window of the nanocomposite during preparation. Simultaneously, it is required to meet requirements such as controllable particle size, low polydispersity index, stable dispersion, stable lyophilized storage, and rapid redispersibility after reconstitution. For oral formulations aimed at preventing acetaminophen-induced liver damage, it is also necessary to further control the residual ethanol content and water content of the lyophilized powder, maintain the integrity of the core and shell structures, and promote the effective release and absorption of 8-O-acetylharbazoside to achieve both preparation feasibility and application feasibility. Meanwhile, the molding compatibility and quality stability of oral formulation raw material powder, granules, capsule contents or oral lyophilized reconstituted formulations also need to be considered. Therefore, how to uniformly handle multiple steps such as compounding, ionic crosslinking, lyophilization and reconstitution in the same nanocomposition has become a key issue in the research and development of this type of oral nanocomposition.

[0003] Currently, various approaches have been proposed for the oral delivery of poorly soluble natural active ingredients, including phospholipid complex systems and chitosan derivative nanocarriers. For example, Chinese patent CN1561992A discloses a pro-liposome formulation containing milk thistle extract and its preparation method, primarily improving storage stability and oral absorption through phospholipids and related excipients. Another example is Chinese patent CN102641245A, which discloses chitosan-chitosan derivative nanospheres loaded with poorly soluble drugs, their preparation method, and their application as oral formulations, emphasizing the loading, release control, and mucosal interaction of chitosan derivative nanospheres with poorly soluble drugs. However, existing research often focuses on single solubilization, single absorption promotion, or single carrier optimization pathways. There is a lack of comprehensive design for how to maintain low viscosity and wide processing window under high loading and solid content conditions, how to balance low ethanol residue, low water content of lyophilized powder, rapid redispersion, and integrity of core and shell structure before and after freeze-drying, and how to unify interfacial composite, ionic crosslinking stability, and oral release absorption into a single industrially feasible system. In particular, there is a lack of systematic solutions for simultaneous optimization of multiple objectives. Summary of the Invention

[0004] The purpose of this invention is to provide an 8-O-acetylharbazoside nanocomposition and its preparation method, which solves the problem that current oral 8-O-acetylharbazoside nanocomposites are difficult to balance in terms of loading capacity, particle size distribution, freeze-drying stability, reconstitution and redispersibility, and release and absorption.

[0005] This invention constructs a core phase using an 8-O-acetylharbazoside-soybean lecithin complex to improve the loading and stability of 8-O-acetylharbazoside. Then, a shell layer is formed by double-modified chitosan with lactobionic acid and thioglycolic acid and sodium tripolyphosphate to coat the core phase. Through ionic crosslinking, it synergistically achieves low viscosity, wide processing window, small particle size, low polydispersity index, freeze-drying stability, resolubilization and redispersibility, and release and absorption.

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

[0007] An 8-O-acetylharbazoside nanocomposition comprises the following components: 8-O-acetylharbazoside-soybean lecithin complex; lactobionic acid and thioglycolic acid-modified chitosan; sodium tripolyphosphate;

[0008] The 8-O-acetylharbazoside-soybean lecithin complex is the core phase, and the lactobionic acid and thioglycolic acid dual-modified chitosan forms a shell layer coating the core phase with the sodium tripolyphosphate. The ratio of the 8-O-acetylharbazoside-soybean lecithin complex, the lactobionic acid and thioglycolic acid dual-modified chitosan, and the sodium tripolyphosphate by weight is 1:0.5-3.0:0.02-0.20.

[0009] The nanocomposition has a particle size of 80-220 nm and a polydispersity index of 0.10-0.30; the lactobionic acid substitution degree of the lactobionic acid and thioglycolic acid dual-modified chitosan is 2-10 mol%, and the thiol content is 30-180 μmol / g; in the nanocomposition, the shell is formed by the lactobionic acid and thioglycolic acid dual-modified chitosan and the sodium tripolyphosphate through ionic crosslinking, and coats the core phase.

[0010] Furthermore, the lactobionic acid and thioglycolic acid dual-modified chitosan is prepared through the following steps:

[0011] A1. Disperse 100 parts by weight of lactobionic acid modified chitosan intermediate in 1000-3000 parts by weight of purified water;

[0012] A2. Adjust the pH of the system to 4.5-6.0 using sodium hydroxide;

[0013] A3. Add 5-40 parts by weight of mercaptoacetic acid, 5-30 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 3-20 parts by weight of N-hydroxysuccinimide to the system obtained in step A2, and react at 20-30°C under nitrogen protection for 2-10 hours.

[0014] A4. Dialyze with purified water for 12-48 hours, then vacuum dry at 35-55°C for 8-24 hours to obtain the lactobionic acid and thioglycolic acid dual-modified chitosan. The thiol content of the lactobionic acid and thioglycolic acid dual-modified chitosan is 30-180 μmol / g, and the residual amount of free thioglycolic acid is not higher than 0.5 wt%.

[0015] Furthermore, the lactobionic acid-modified chitosan intermediate is prepared through the following steps:

[0016] B1. Add 100 parts by weight of chitosan to 1000-3000 parts by weight of a 0.5-2.0 vol% aqueous solution of acetic acid prepared by acetic acid and purified water, stir and disperse to obtain a chitosan dispersion system;

[0017] B2. Adjust the pH of the chitosan dispersion system to 4.5-5.5 using sodium hydroxide;

[0018] B3. Add 10-60 parts by weight of lactobionic acid, 8-40 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 5-25 parts by weight of N-hydroxysuccinimide to the system obtained in step B2, and react at 20-35°C for 4-12 hours.

[0019] B4. Dialyze with purified water for 12-48 hours, then vacuum dry at 40-60°C for 8-24 hours to obtain the lactobionic acid modified chitosan intermediate, wherein the lactobionic acid degree of substitution of the lactobionic acid modified chitosan intermediate is 2-10 mol.

[0020] Furthermore, the dialysis described in step B4 uses a dialysis bag with a molecular weight cutoff of 3000-14000 Da, and is performed in purified water for 12-48 hours, with the purified water being replaced every 4-12 hours.

[0021] Furthermore, the 8-O-acetylharbazoside-soybean lecithin complex is prepared by the following steps:

[0022] C1. Add 10-50 parts by weight of 8-O-acetylharbazoside and soybean lecithin to 300-3000 parts by weight of ethanol, wherein the mass ratio of 8-O-acetylharbazoside to soybean lecithin is 1:1-1:6;

[0023] C2. Stir at 40-60℃ for 0.5-4 hours;

[0024] C3. After removing ethanol under reduced pressure at 35-45℃, dry under vacuum at 25-40℃ for 4-12 hours.

[0025] C4. The 8-O-acetylharbazoside-soybean lecithin complex is obtained, wherein the residual ethanol content in the complex is not higher than 0.5 wt%, and the loading of 8-O-acetylharbazoside in the complex is 10-50 wt%.

[0026] As a concept of this invention, an 8-O-acetylharbazoside-soybean lecithin complex is synergistically constructed with lactobionic acid and thioglycolic acid-modified chitosan / sodium tripolyphosphate to enhance the oral delivery performance of 8-O-acetylharbazoside. By first establishing a composite core phase of 8-O-acetylharbazoside and soybean lecithin, a foundation for higher loading and subsequent dispersion can be provided while ensuring compatibility. Then, the shell layer formed by the lactobionic acid and thioglycolic acid-modified chitosan and sodium tripolyphosphate coats the core phase, simultaneously constraining particle size growth, reducing the polydispersity index, and maintaining stable nano-dispersion. This structure also facilitates maintaining the integrity of the composition during freeze-drying, storage, and reconstitution, while simultaneously achieving low ethanol residue, low water content in the freeze-dried powder, rapid redispersibility, and oral release and absorption, thus alleviating the problem that existing 8-O-acetylharbazoside phospholipid composite nanosystems struggle to achieve multiple objectives simultaneously.

[0027] This invention also discloses a method for preparing an 8-O-acetylharbazoside nanocomposition, comprising the following steps:

[0028] S1. Add 100 parts by weight of lactobionic acid and thioglycolic acid dual-modified chitosan to 1000-5000 parts by weight of 0.2-1.0 vol% acetic acid aqueous solution prepared by acetic acid and purified water to obtain the shell phase of lactobionic acid and thioglycolic acid dual-modified chitosan;

[0029] S2. The 8-O-acetylharbazoside-soybean lecithin complex is added to the shell phase and dispersed at 3000-12000 r / min for 3-15 min to obtain a dispersion system, wherein the mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex to the lactobionic acid and thioglycolic acid dual-modified chitosan is 1:0.5-3.0;

[0030] S3. Dissolve sodium tripolyphosphate in purified water to obtain an aqueous solution of sodium tripolyphosphate. Add the aqueous solution of sodium tripolyphosphate dropwise to the dispersion system at a temperature of 20-30℃ and a pH of 4.5-6.5, and continue stirring for 10-60 minutes to carry out ionic cross-linking. The mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex, the lactobionic acid and thioglycolic acid-modified chitosan, and the sodium tripolyphosphate is 1:0.5-3.0:0.02-0.20.

[0031] S4. Subsequently, freeze-drying was performed to obtain the 8-O-acetylharbazoside nanocomposition.

[0032] Furthermore, in step S2, the dispersion speed is 5000-10000 r / min, and the dispersion time is 5-10 min; in step S3, the dropping time of the sodium tripolyphosphate aqueous solution is 15-40 min, the ion crosslinking temperature is 20-25℃, and the pH value of the ion crosslinking system is 5.0-6.0.

[0033] Further, in step S4, based on the total mass of the 8-O-acetylharbazoside-soybean lecithin complex, the lactobionic acid and thioglycolic acid double-modified chitosan, and the sodium tripolyphosphate, a lyophilization protectant of 5-30 wt% is added. The lyophilization protectant is selected from one or both of D-trehalose dihydrate and D-mannitol. The pre-freezing temperature is -40℃ to -20℃, the pre-freezing time is 2-12 h, the main drying pressure is 0.00002-0.00010 MPa, and the main drying time is 12-36 h.

[0034] Furthermore, the residual ethanol content in the obtained 8-O-acetylharbazoside nanocomposition is no higher than 0.5 wt%, and the water content of the lyophilized powder is no higher than 5 wt%.

[0035] Furthermore, after step S4, a formulation forming step is also included, wherein the obtained 8-O-acetylharbazoside nanocomposition is made into oral formulation raw material powder, granules, capsule contents, tablet granule intermediates, oral suspensions, or oral lyophilized reconstituted formulations; when an oral formulation with a unit dose is prepared, the unit dose is 1-100 mg based on 8-O-acetylharbazoside.

[0036] Furthermore, the application of the 8-O-acetylharbazoside nanocomposition in the preparation of a drug for preventing acetaminophen-induced liver injury, wherein the drug is an oral formulation selected from capsules, tablets, granules, oral suspensions and oral lyophilized reconstituted formulations, wherein the unit dose of the oral formulation, calculated as 8-O-acetylharbazoside, is 5-50 mg.

[0037] Furthermore, the pH value of the system is adjusted using an aqueous sodium hydroxide solution with a concentration of 0.1-2.0 mol / L, and the solution is added dropwise.

[0038] Furthermore, nitrogen protection is used in the mercaptoacetic acid modification step. The nitrogen protection includes replacing the air in the reaction vessel with nitrogen 2-5 times before the reaction and / or continuously introducing nitrogen during the reaction.

[0039] Furthermore, the ethanol mentioned in step C1 is anhydrous ethanol or ethanol with a volume fraction of 95 vol%.

[0040] Furthermore, the vacuum degree for depressurization removal of ethanol and vacuum drying in step C3 is 0.001-0.01 MPa.

[0041] Furthermore, the sodium tripolyphosphate is dissolved in 100-1000 times its weight of purified water.

[0042] Furthermore, the sodium tripolyphosphate aqueous solution is added dropwise to the dispersion system at a rate of 0.5-5.0 mL / min.

[0043] Furthermore, the freeze-drying includes pre-freezing at -40℃ to -20℃ for 2-12 hours, and main drying at -30℃ to -10℃ under 0.00002-0.00010 MPa for 12-36 hours.

[0044] Furthermore, the particle size and polydispersity index of the nanocomposite were determined by dynamic light scattering at a test temperature of 25°C and the dispersion medium was purified water or phosphate buffered saline (PBS).

[0045] Furthermore, the degree of lactobionic acid substitution was determined by titration or nuclear magnetic resonance, the thiol content was determined by the Ellman reagent method, and the 8-O-acetylharbazoside loading was determined by high performance liquid chromatography.

[0046] Furthermore, the residual ethanol content was determined by headspace gas chromatography, the water content of the lyophilized powder was determined by Karl Fischer method, and the residual free thioglycolic acid content was determined by high performance liquid chromatography.

[0047] Furthermore, the core phase and shell structure of the nanocomposite were characterized by transmission electron microscopy, zeta potential change, or encapsulation efficiency measurement.

[0048] Furthermore, the efficacy of the drug for preventing acetaminophen-induced liver injury was evaluated using animal models, and the evaluation indicators included ALT, AST, liver tissue pathology, and oxidative stress indicators.

[0049] As another aspect of this invention, the present invention employs a preparation path that involves first preparing a chitosan shell phase modified with lactobionic acid and thioglycolic acid, then dispersing an 8-O-acetylharbazoside-soybean lecithin complex, followed by the dropwise addition of sodium tripolyphosphate for ionic crosslinking and freeze-drying with a freeze-drying protectant. This approach primarily enhances the process controllability and molding stability of the nanocomposition. This method implements core phase formation, shell construction, and freeze-drying stepwise, which facilitates control over dispersion speed, dropwise addition process, ionic crosslinking conditions, and pre-freezing and main drying processes. This allows the nanocomposition to achieve both low viscosity and a wide processing window during the preparation stage, and low ethanol residue, low water content in the freeze-dried powder, and rapid redispersibility after reconstitution during the finished product stage. It also provides a stable foundation for subsequent preparation of oral formulation raw materials, granules, capsule contents, tablet granule intermediates, oral suspensions, or oral freeze-dried reconstituted formulations.

[0050] The 8-O-acetylharbazoside-soybean lecithin complex focuses on providing a stable core phase and high loading capacity for 8-O-acetylharbazoside, improving its composite state in nanocomposites, and creating conditions for subsequent release and absorption. The lactobionic acid and thioglycolic acid-modified chitosan focuses on forming a shell coating the core phase with sodium tripolyphosphate, stabilizing the dispersion interface, constraining particle size, and reducing the polydispersity index through ionic crosslinking, while simultaneously enhancing structure retention during lyophilization and reconstitution. The synergistic effect of these two components is that, on the one hand, the core phase helps maintain drug composite stability and effective release; on the other hand, the shell helps maintain stable nanodispersion and reduce the risk of aggregation and instability. Therefore, it can simultaneously improve processing window, storage stability, and oral absorption.

[0051] Beneficial technical effects

[0052] 1. By pre-constructing the core phase with soybean lecithin and then using a chitosan / sodium tripolyphosphate double-modified structure with lactobionic acid and thioglycolic acid, this invention can balance the composite stability of 8-O-acetylharbazoside with the interfacial stability of the nanocomposite, which is beneficial to obtaining nanocomposites with concentrated particle size and stable dispersion on the basis of high loading and solid content.

[0053] 2. By constructing a confined shell through ionic crosslinking and coordinating the control of dispersion speed, dropping process and pH value, this invention can reduce the risk of aggregation and instability during the preparation process, and achieve a better balance between low viscosity, wide processing window and batch consistency. Compared with a single composite or single nano-scale path, it is more conducive to subsequent scale-up preparation.

[0054] 3. By controlling the residual amount of ethanol, the residual amount of free mercaptoacetic acid, the lyophilization protectant, and the lyophilization conditions, this invention helps to reduce the residue and water content, improve the storage stability of the lyophilized powder, and quickly restore the nano-dispersed state after reconstitution, thereby reducing the adverse effects of structural collapse, aggregation, and performance degradation on oral application.

[0055] 4. Through the synergistic regulation of the core phase and shell, the present invention can maintain the structural integrity of the nanocomposite and facilitate its release and absorption in an oral environment, thereby providing a more feasible oral formulation basis for 8-O-acetylharbazoside to prevent acetaminophen-induced liver injury, and demonstrating better comprehensive adaptability than existing single-carrier optimization schemes. Attached Figure Description

[0056] Figure 1 This is an overlay of the DLS particle size distribution of Example 1 and Comparative Example 10.

[0057] Figure 2 The particle size recovery curves after resolution are shown for Examples 1, 7, and 10.

[0058] Figure 3 Time-particle size diagrams for Example 1, Comparative Example 7, and Comparative Example 10.

[0059] Figure 4 The images are FTIR overlays of Example 1, Comparative Example 8, and Comparative Example 9.

[0060] Figure 5 The in vitro release curves are for Example 1, Comparative Example 8, and Comparative Example 9.

[0061] Figure 6 a is a low-magnification scanning electron microscope image of the 8-O-acetylharbazoside nanocomposition prepared in Example 1.

[0062] Figure 6 b is a high-magnification scanning electron microscope image of the 8-O-acetylharbazoside nanocomposition prepared in Example 1.

[0063] Figure 7 a is a bright-field transmission electron microscopy image of the 8-O-acetylharbazoside nanocomposition prepared in Example 1.

[0064] Figure 7 b is a partial transmission electron microscope (TEM) magnification of the 8-O-acetylharbazoside nanocomposition prepared in Example 1.

[0065] Figure 7 c is a high-resolution transmission electron microscope image of the 8-O-acetylharbazoside nanocomposite prepared in Example 1.

[0066] Figure 8 Macroscopic photograph of the lyophilized powder of the 8-O-acetylharbazoside nanocomposition prepared in Example 1. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0068] Example 1

[0069] I. Preparation of Lactobionic Acid-Modified Chitosan Intermediate: 100g of chitosan was added to 2000g of a 1.2 vol% acetic acid aqueous solution prepared from acetic acid and purified water, and the mixture was stirred thoroughly to obtain a chitosan dispersion system. The pH of the chitosan dispersion system in this embodiment was adjusted to 5.0 by dropwise addition of a 0.5 mol / L sodium hydroxide aqueous solution. 35g of lactobionic acid, 24g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 15g of N-hydroxysuccinimide were added to the pH-adjusted system, and the reaction was carried out at 28°C for 8 hours. After the reaction was completed, the mixture was dialyzed in purified water for 30 hours using a dialysis bag with a molecular weight cutoff of 8000 Da, and the purified water was replaced every 8 hours. After dialysis, the mixture was vacuum dried at 50°C for 16 hours to obtain the lactobionic acid-modified chitosan intermediate of this embodiment. The degree of lactobionic acid substitution of the lactobionic acid-modified chitosan intermediate in this embodiment was 6 mol%.

[0070] II. Preparation of chitosan modified with lactobionic acid and thioglycolic acid: 100g of the lactobionic acid-modified chitosan intermediate prepared above was dispersed in 2000g of purified water. The pH of the system was adjusted to 5.2 by dropwise addition of a 1.0mol / L sodium hydroxide aqueous solution. 22g of thioglycolic acid, 18g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 11g of N-hydroxysuccinimide were added to the pH-adjusted system. The air in the reaction vessel was purged with nitrogen three times before the reaction. The reaction was carried out at 25℃ for 6 hours under nitrogen protection, with nitrogen continuously purging during the reaction. After the reaction was completed, the mixture was dialyzed in purified water for 30 hours using a dialysis bag with a molecular weight cutoff of 8000 Da, with the purified water replaced every 8 hours. After dialysis, the chitosan was vacuum dried at 45°C for 16 hours to obtain the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. The thiol content of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment is 100 μmol / g, the residual amount of free thioglycolic acid is 0.2 wt%, and the degree of lactobionic acid substitution is 6 mol.

[0071] III. Preparation of the 8-O-acetylharbazoside-soybean lecithin complex: 2.5 g of 8-O-acetylharbazoside and 7.5 g of soybean lecithin were added to 200 g of 95 vol% ethanol. In this example, the mass ratio of 8-O-acetylharbazoside to soybean lecithin was 1:3. The mixture was stirred at 50 °C for 2 h. After stirring, the ethanol was removed under reduced pressure at 40 °C and a vacuum degree of 0.005 MPa, and then vacuum dried at 33 °C and a vacuum degree of 0.005 MPa for 8 h. The 8-O-acetylharbazoside-soybean lecithin complex of this example was obtained. The residual ethanol content in the complex of this example was 0.15 wt%, and the loading of 8-O-acetylharbazoside in the complex of this example was 25 wt%.

[0072] IV. Preparation of the 8-O-acetylharbazoside nanocomposition: 7.5g of the lactobionic acid and thioglycolic acid dual-modified chitosan prepared above was added to 150g of a 0.6 vol% acetic acid aqueous solution prepared from acetic acid and purified water to obtain the shell phase of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. 5g of the 8-O-acetylharbazoside-soybean lecithin complex prepared above was added to the shell phase of this embodiment and dispersed at 7000 r / min for 8 min to obtain a dispersion system. In this embodiment, the mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex to the lactobionic acid and thioglycolic acid dual-modified chitosan was 1:1.5. 0.5g of sodium tripolyphosphate was dissolved in 50g of purified water to obtain a sodium tripolyphosphate aqueous solution. In this embodiment, the sodium tripolyphosphate was dissolved in 100 times its mass of purified water. The pH of the dispersion system was adjusted to 5.5 by dropwise addition of a 0.8 mol / L sodium hydroxide aqueous solution. At a temperature of 23°C, an aqueous solution of sodium tripolyphosphate was added dropwise to the dispersion system of this embodiment at a rate of 2.5 mL / min for 20 min. Stirring was continued for 35 min to perform ionic crosslinking. In this embodiment, the mass ratio of 8-O-acetylgabaside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate was 1:1.5:0.10. After ionic crosslinking, based on the total mass of the 8-O-acetylgabaside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate, 18 wt% D-trehalose dihydrate was added as a lyophilization protectant. The mixture was pre-frozen at -30°C for 7 h, and then primarily dried at -20°C under 0.00006 MPa pressure for 24 h to obtain the 8-O-acetylgabaside nanocomposition of this embodiment. The residual ethanol content in the nanocomposition of this embodiment was 0.12 wt%, and the water content of the lyophilized powder was 2.8 wt%. The nanocomposition of this embodiment has a particle size of 150 nm and a polydispersity index of 0.20. The nanocomposition of this embodiment has a core-shell structure, wherein the 8-O-acetylharbazoside-soybean lecithin complex is the core phase, and the chitosan double-modified by lactobionic acid and thioglycolic acid and sodium tripolyphosphate are ionically crosslinked to form a shell layer that coats the core phase.

[0073] V. Formulation: The 8-O-acetylharbazoside nanocomposition prepared above is formulated into capsules. The unit dose is 30 mg based on 8-O-acetylharbazoside. The capsule formulation of this embodiment is used to prepare an oral medication for preventing acetaminophen-induced liver injury.

[0074] This embodiment is suitable for large-scale production scenarios that require stable process parameters and batch consistency. It is particularly suitable as a standard process for industrial scale-up and can be widely used in the fields of liver injury prevention and intervention and oral formulation development.

[0075] Example 2

[0076] I. Preparation of Lactobionic Acid-Modified Chitosan Intermediate: 100g of chitosan was added to 1500g of a 0.8 vol% acetic acid aqueous solution prepared from acetic acid and purified water, and the mixture was stirred thoroughly to obtain a chitosan dispersion system. The pH of the chitosan dispersion system in this embodiment was adjusted to 4.7 by dropwise addition of a 0.3 mol / L sodium hydroxide aqueous solution. 20g of lactobionic acid, 15g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 10g of N-hydroxysuccinimide were added to the pH-adjusted system, and the reaction was carried out at 24°C for 6 hours. After the reaction was completed, the mixture was dialyzed in purified water for 20 hours using a dialysis bag with a molecular weight cutoff of 5000 Da, and the purified water was replaced every 5 hours. After dialysis, the mixture was vacuum dried at 45°C for 12 hours to obtain the lactobionic acid-modified chitosan intermediate of this embodiment. The degree of lactobionic acid substitution of the lactobionic acid-modified chitosan intermediate in this embodiment was 4 mol%.

[0077] II. Preparation of chitosan modified with lactobionic acid and thioglycolic acid: 100g of the lactobionic acid-modified chitosan intermediate prepared above was dispersed in 1500g of purified water. The pH of the system was adjusted to 4.8 by dropwise addition of a 0.6mol / L sodium hydroxide aqueous solution. 12g of thioglycolic acid, 10g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 6g of N-hydroxysuccinimide were added to the pH-adjusted system. Before the reaction, the air in the reaction vessel was replaced with nitrogen four times. The reaction was carried out at 22℃ for 4h under nitrogen protection, with nitrogen continuously purging during the reaction. After the reaction was completed, the mixture was dialyzed in purified water for 24h using a dialysis bag with a molecular weight cutoff of 5000Da, and the purified water was replaced every 6h. After dialysis, the chitosan was vacuum dried at 40°C for 12 hours to obtain the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. The thiol content of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment is 60 μmol / g, the residual amount of free thioglycolic acid is 0.15 wt%, and the degree of lactobionic acid substitution is 4 mol.

[0078] III. Preparation of the 8-O-acetylharbazoside-soybean lecithin complex: 3g of 8-O-acetylharbazoside and 6g of soybean lecithin were added to 180g of anhydrous ethanol. In this example, the mass ratio of 8-O-acetylharbazoside to soybean lecithin was 1:2. The mixture was stirred at 45°C for 1.5h. After stirring, the ethanol was removed under reduced pressure at 38°C and a vacuum of 0.003MPa, and then vacuum dried at 30°C and a vacuum of 0.003MPa for 6h. The 8-O-acetylharbazoside-soybean lecithin complex of this example was obtained. The residual ethanol content in the complex of this example was 0.18wt%, and the loading of 8-O-acetylharbazoside in the complex of this example was 33.3wt%.

[0079] IV. Preparation of the 8-O-acetylharbazoside nanocomposition: 4.8g of the lactobionic acid and thioglycolic acid dual-modified chitosan prepared above was added to 120g of a 0.4 vol% acetic acid aqueous solution prepared from acetic acid and purified water to obtain the shell phase of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. 6g of the 8-O-acetylharbazoside-soybean lecithin complex prepared above was added to the shell phase of this embodiment and dispersed at 6000 r / min for 6 min to obtain a dispersion system. In this embodiment, the mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex to the lactobionic acid and thioglycolic acid dual-modified chitosan was 1:0.8. 0.3g of sodium tripolyphosphate was dissolved in 90g of purified water to obtain a sodium tripolyphosphate aqueous solution. In this embodiment, the sodium tripolyphosphate was dissolved in 300 times its mass of purified water. The pH of the dispersion system was adjusted to 5.2 by dropwise addition of a 0.5 mol / L sodium hydroxide aqueous solution. At a temperature of 22°C, an aqueous solution of sodium tripolyphosphate was added dropwise to the dispersion system of this embodiment at a rate of 3.0 mL / min for 30 min. Stirring was continued for 25 min to perform ionic crosslinking. In this embodiment, the mass ratio of 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate was 1:0.8:0.05. After ionic crosslinking, based on the total mass of the 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate, 12 wt% D-mannitol was added as a lyophilization protectant. The mixture was pre-frozen at -25°C for 5 h, and then primarily dried at -15°C for 18 h under a pressure of 0.00004 MPa to obtain the 8-O-acetylharbazoside nanocomposition of this embodiment. The residual ethanol content in the nanocomposition of this embodiment was 0.10 wt%, and the water content of the lyophilized powder was 2.2 wt%. The nanocomposition of this embodiment has a particle size of 100 nm and a polydispersity index of 0.15. The nanocomposition of this embodiment has a core-shell structure, wherein the 8-O-acetylharbazoside-soybean lecithin complex is the core phase, and the chitosan double-modified by lactobionic acid and thioglycolic acid and sodium tripolyphosphate are ionically crosslinked to form a shell layer that coats the core phase.

[0080] V. Formulation: The 8-O-acetylharbazoside nanocomposition prepared above is formulated into an oral suspension. The unit dose is 15 mg based on 8-O-acetylharbazoside. This oral suspension is used to prepare an oral medication for preventing acetaminophen-induced liver injury.

[0081] This embodiment is suitable for drug delivery scenarios that require rapid absorption and high bioavailability. The small particle size is beneficial to improving oral absorption efficiency, and the higher drug load can reduce the dosage. It is particularly suitable for early prevention and intervention of acute liver injury, as well as for the development of formulations with special requirements for drug particle size and load.

[0082] Example 3

[0083] I. Preparation of Lactobionic Acid-Modified Chitosan Intermediate: 100g of chitosan was added to 2500g of a 1.7 vol% acetic acid aqueous solution prepared from acetic acid and purified water, and the mixture was stirred thoroughly to obtain a chitosan dispersion system. The pH of the chitosan dispersion system in this embodiment was adjusted to 5.3 by dropwise addition of a 1.5 mol / L sodium hydroxide aqueous solution. 50g of lactobionic acid, 32g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 20g of N-hydroxysuccinimide were added to the pH-adjusted system, and the reaction was carried out at 32°C for 10h. After the reaction was completed, the mixture was dialyzed in purified water for 40h using a dialysis bag with a molecular weight cutoff of 12000 Da, and the purified water was replaced every 10h. After dialysis, the mixture was vacuum dried at 56°C for 20h to obtain the lactobionic acid-modified chitosan intermediate of this embodiment. The degree of lactobionic acid substitution of the lactobionic acid-modified chitosan intermediate in this embodiment was 8 mol%.

[0084] II. Preparation of chitosan modified with lactobionic acid and thioglycolic acid: 100g of the lactobionic acid-modified chitosan intermediate prepared above was dispersed in 2600g of purified water. The pH of the system was adjusted to 5.8 by dropwise addition of a 1.6mol / L sodium hydroxide aqueous solution. 34g of thioglycolic acid, 25g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 17g of N-hydroxysuccinimide were added to the pH-adjusted system. The air in the reaction vessel was purged with nitrogen five times before the reaction. The reaction was carried out at 28℃ for 8 hours under nitrogen protection, with nitrogen continuously purging during the reaction. After the reaction was completed, the mixture was dialyzed in purified water for 42 hours using a dialysis bag with a molecular weight cutoff of 12000 Da, with the purified water replaced every 10 hours. After dialysis, the chitosan was vacuum dried at 52°C for 20 hours to obtain the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. The thiol content of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment is 140 μmol / g, the residual amount of free thioglycolic acid is 0.28 wt%, and the degree of lactobionic acid substitution is 8 mol.

[0085] III. Preparation of the 8-O-acetylharbazoside-soybean lecithin complex: 1.6 g of 8-O-acetylharbazoside and 8 g of soybean lecithin were added to 240 g of 95 vol% ethanol. In this example, the mass ratio of 8-O-acetylharbazoside to soybean lecithin was 1:5. The mixture was stirred at 56 °C for 3 h. After stirring, the ethanol was removed under reduced pressure at 43 °C and a vacuum of 0.008 MPa, and then vacuum dried at 38 °C and a vacuum of 0.008 MPa for 10 h. The 8-O-acetylharbazoside-soybean lecithin complex of this example was obtained. The residual ethanol content in the complex of this example was 0.32 wt%, and the loading of 8-O-acetylharbazoside in the complex of this example was 16.7 wt%.

[0086] IV. Preparation of the 8-O-acetylharbazoside nanocomposition: 10g of the lactobionic acid and thioglycolic acid dual-modified chitosan prepared above was added to 200g of a 0.9 vol% acetic acid aqueous solution prepared from acetic acid and purified water to obtain the shell phase of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. 4g of the 8-O-acetylharbazoside-soybean lecithin complex prepared above was added to the shell phase of this embodiment and dispersed at 9000 r / min for 10 min to obtain a dispersion system. In this embodiment, the mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex to the lactobionic acid and thioglycolic acid dual-modified chitosan was 1:2.5. 0.64g of sodium tripolyphosphate was dissolved in 128g of purified water to obtain a sodium tripolyphosphate aqueous solution. In this embodiment, the sodium tripolyphosphate was dissolved in 200 times its mass of purified water. The pH of the dispersion system was adjusted to 5.8 by dropwise addition of a 1.2 mol / L sodium hydroxide aqueous solution. At a temperature of 24°C, an aqueous solution of sodium tripolyphosphate was added dropwise to the dispersion system of this embodiment at a rate of 4.0 mL / min for 32 min. Stirring was continued for 50 min to perform ionic crosslinking. In this embodiment, the mass ratio of 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate was 1:2.5:0.16. After ionic crosslinking, based on the total mass of the 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate, 24 wt% of a lyophilization protectant was added. In this embodiment, the lyophilization protectant was a mixture of D-trehalose dihydrate and D-mannitol, with D-trehalose dihydrate accounting for 60% and D-mannitol accounting for 40%. The nanocomposition of this embodiment was obtained by pre-freezing at -35℃ for 10 h, followed by primary drying at -25℃ under 0.00008 MPa pressure for 32 h. The residual ethanol content in the nanocomposition of this embodiment was 0.22 wt%, and the water content of the lyophilized powder was 3.8 wt%. The particle size of the nanocomposition of this embodiment was 190 nm, and the polydispersity index was 0.25. The nanocomposition of this embodiment has a core-shell structure, wherein the 8-O-acetylharbazoside-soybean lecithin complex is the core phase, and the chitosan double-modified with lactobionic acid and thioglycolic acid is ionically cross-linked with sodium tripolyphosphate to form a shell coating the core phase.

[0087] V. Formulation: The 8-O-acetylharbazoside nanocomposition prepared above is formulated into tablet granule intermediates. The unit dose is 40 mg based on 8-O-acetylharbazoside. The tablets of this embodiment are used to prepare an oral medication for preventing acetaminophen-induced liver injury.

[0088] This embodiment is suitable for drug delivery scenarios that require long-acting sustained release and enhanced mucosal retention. The thicker shell structure and higher thiol content are beneficial for improving the adhesion ability of the gastrointestinal mucosa and prolonging the retention time of the drug in the body. It is particularly suitable for continuous prevention of chronic liver injury and treatment regimens that require prolonged action time, as well as storage and transportation conditions that require high formulation stability.

[0089] Example 4

[0090] I. Preparation of Lactobionic Acid-Modified Chitosan Intermediate: 100g of chitosan was added to 2800g of a 1.85 vol% acetic acid aqueous solution prepared from acetic acid and purified water, and the mixture was stirred thoroughly to obtain a chitosan dispersion system. The pH of the chitosan dispersion system in this embodiment was adjusted to 5.4 by dropwise addition of a 1.8 mol / L sodium hydroxide aqueous solution. 56g of lactobionic acid, 37g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 23g of N-hydroxysuccinimide were added to the pH-adjusted system, and the reaction was carried out at 33°C for 11h. After the reaction was completed, the mixture was dialyzed in purified water for 44h using a dialysis bag with a molecular weight cutoff of 13000 Da, and the purified water was replaced every 11h. After dialysis, the mixture was vacuum dried at 58°C for 22h to obtain the lactobionic acid-modified chitosan intermediate of this embodiment. The degree of lactobionic acid substitution of the lactobionic acid-modified chitosan intermediate in this embodiment was 9 mol%.

[0091] II. Preparation of chitosan modified with lactobionic acid and thioglycolic acid: 100g of the lactobionic acid-modified chitosan intermediate prepared above was dispersed in 2850g of purified water. The pH of the system was adjusted to 5.9 by dropwise addition of a 1.85mol / L sodium hydroxide aqueous solution. 37g of thioglycolic acid, 28g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 19g of N-hydroxysuccinimide were added to the pH-adjusted system. The air in the reaction vessel was purged with nitrogen five times before the reaction. The reaction was carried out at 29℃ for 9 hours under nitrogen protection, with nitrogen continuously purging during the reaction. After the reaction was completed, the mixture was dialyzed in purified water for 46 hours using a dialysis bag with a molecular weight cutoff of 13000 Da, with the purified water replaced every 11 hours. After dialysis, the chitosan was vacuum dried at 53°C for 22 hours to obtain the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. The thiol content of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment is 165 μmol / g, the residual amount of free thioglycolic acid is 0.38 wt%, and the degree of lactobionic acid substitution is 9 mol.

[0092] III. Preparation of the 8-O-acetylharbazoside-soybean lecithin complex: 1.4 g of 8-O-acetylharbazoside and 7.7 g of soybean lecithin were added to 228 g of anhydrous ethanol. In this example, the mass ratio of 8-O-acetylharbazoside to soybean lecithin was 1:5.5. The mixture was stirred at 58 °C for 3.5 h. After stirring, the ethanol was removed under reduced pressure at 44 °C and a vacuum degree of 0.009 MPa, and then vacuum dried at 39 °C and a vacuum degree of 0.009 MPa for 11 h. The 8-O-acetylharbazoside-soybean lecithin complex of this example was obtained. The residual ethanol content in the complex of this example was 0.42 wt%, and the loading of 8-O-acetylharbazoside in the complex of this example was 15.4 wt%.

[0093] IV. Preparation of the 8-O-acetylharbazoside nanocomposition: 9.8g of the lactobionic acid and thioglycolic acid dual-modified chitosan prepared above was added to 196g of a 0.92 vol% acetic acid aqueous solution prepared from acetic acid and purified water to obtain the shell phase of the lactobionic acid and thioglycolic acid dual-modified chitosan of this embodiment. 3.5g of the 8-O-acetylharbazoside-soybean lecithin complex prepared above was added to the shell phase of this embodiment and dispersed at 9500 r / min for 9 min to obtain a dispersion system. In this embodiment, the mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex to the lactobionic acid and thioglycolic acid dual-modified chitosan was 1:2.8. 0.63g of sodium tripolyphosphate was dissolved in 63g of purified water to obtain a sodium tripolyphosphate aqueous solution. In this embodiment, the sodium tripolyphosphate was dissolved in 100 times its mass of purified water. The pH of the dispersion system was adjusted to 5.9 by dropwise addition of a 1.75 mol / L sodium hydroxide aqueous solution. At a temperature of 24°C, an aqueous solution of sodium tripolyphosphate was added dropwise to the dispersion system of this embodiment at a rate of 3.0 mL / min for 21 min. Stirring was continued for 55 min to perform ionic crosslinking. In this embodiment, the mass ratio of 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate was 1:2.8:0.18. After ionic crosslinking, based on the total mass of the 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate, 27 wt% of a lyophilization protectant was added. In this embodiment, the lyophilization protectant was a mixture of D-trehalose dihydrate and D-mannitol, with D-trehalose dihydrate accounting for 70% and D-mannitol accounting for 30%. The nanocomposition of 8-O-acetylharbazoside was obtained by pre-freezing at -38℃ for 11 h and then main drying at -28℃ for 34 h under a pressure of 0.000092 MPa. The residual ethanol content in the nanocomposition of this embodiment was 0.28 wt%, and the water content of the lyophilized powder was 4.5 wt%. The particle size of the nanocomposition of this embodiment was 210 nm, and the polydispersity index was 0.12. The nanocomposition of this embodiment has a core-shell structure, wherein the 8-O-acetylharbazoside-soybean lecithin complex is the core phase, and the chitosan modified with lactobionic acid and thioglycolic acid and sodium tripolyphosphate are ionically crosslinked to form a shell coating the core phase.

[0094] V. Formulation: The 8-O-acetylharbazoside nanocomposition prepared above is formulated into an oral lyophilized reconstituted formulation. The unit dose is 48 mg based on 8-O-acetylharbazoside. This oral lyophilized reconstituted formulation is used to prepare an oral medication for preventing acetaminophen-induced liver injury.

[0095] This embodiment is suitable for precision drug delivery scenarios where high uniformity of formulation and mucosal targeting are required. The high degree of modification and high thiol content endow the nanocomposite with excellent mucosal penetration and liver targeting. The low polydispersity index ensures high consistency of quality between batches. It is particularly suitable for long-term preventive medication for high-risk groups of liver injury, as well as clinical research and personalized dosing regimens that require precise control of pharmacokinetic characteristics.

[0096] Comparative Example 1: Basically the same as Example 1, except that in step three, soybean lecithin was replaced with egg yolk lecithin, the mass ratio of 8-O-acetylharbazoside to egg yolk lecithin was still 1:3, and other conditions remained unchanged.

[0097] Comparative Example 2: It is basically the same as Example 1, except that the mass ratio of 8-O-acetylharbazoside to soybean lecithin in step 3 is adjusted to 1:6.5, while other conditions remain unchanged.

[0098] Comparative Example 3: It is basically the same as Example 1, except that in step four, the mass ratio of 8-O-acetylharbazoside-soybean lecithin complex to lactobionic acid and thioglycolic acid-modified chitosan is adjusted to 1:0.3, while other conditions remain unchanged.

[0099] Comparative Example 4: Basically the same as Example 1, except that in step four, the mass ratio of 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid double-modified chitosan and sodium tripolyphosphate was adjusted to 1:1.5:0.25, while other conditions remained unchanged.

[0100] Comparative Example 5: It is basically the same as Example 1, except that the dispersion speed in step four is adjusted to 2000 r / min, the dispersion time is still 8 min, and other conditions remain unchanged.

[0101] Comparative Example 6: Basically the same as Example 1, except that the dropping rate of sodium tripolyphosphate aqueous solution in step four was adjusted to 6.0 mL / min, while other conditions remained unchanged.

[0102] Comparative Example 7: It is basically the same as Example 1, except that in step four, the freeze-drying protectant is still D-trehalose dihydrate, but the amount added is adjusted to 2wt%, and other conditions remain unchanged.

[0103] Comparative Example 8: Essentially the same as Example 1, except that the thioglycolic acid modification step was removed. In step two, thioglycolic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were not added. The lactobionic acid-modified chitosan intermediate obtained in the first part of Example 1 was used directly as the shell material, with other conditions remaining unchanged. This comparative example was used to verify the synergistic effect of lactobionic acid substitution and thioglycolic acid modification.

[0104] Comparative Example 9: Essentially the same as Example 1, except that only thioglycolic acid modification was retained without the addition of lactobionic acid. The thioglycolic acid-modified chitosan was prepared via the following steps: 100g of chitosan was dispersed in 2000g of a 1.2 vol% acetic acid aqueous solution prepared from acetic acid and purified water. The pH was adjusted to 5.2 by dropwise addition of 0.5 mol / L sodium hydroxide aqueous solution. Then, 22g of thioglycolic acid, 18g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 11g of N-hydroxysuccinimide were added. The reaction was carried out at 25°C for 6 hours under nitrogen protection. Subsequently, the mixture was dialyzed in purified water for 30 hours using a dialysis bag with a molecular weight cutoff of 8000 Da, with the purified water being replaced every 8 hours. Finally, the mixture was vacuum dried at 45°C for 16 hours, with other conditions remaining unchanged. This comparative example was used to verify the synergistic effect of lactobionic acid substitution and thioglycolic acid modification.

[0105] Comparative Example 10: Essentially the same as Example 1, except that the step of adding sodium tripolyphosphate aqueous solution dropwise for ionic crosslinking was omitted in step four. Sodium tripolyphosphate was not added. The dispersion system was stirred at 23°C for 35 minutes, and then 18 wt% D-trehalose dihydrate was directly added. The system was then freeze-dried under the pre-freezing and main drying conditions of Example 1, with other conditions remaining unchanged. This comparative example was used to verify the synergistic effect of core-phase coating and ionic crosslinking interface construction.

[0106] This invention employs Fourier transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy to characterize the 8-O-acetylharbazoside nanocomposition and related shell materials prepared in Example 1. Specific results are shown in [the table below]. Figure 4 , Figure 6 and Figure 7 .

[0107] Performance testing:

[0108] The particle size and polydispersity index (PDI) of the lyophilized reconstituted 8-O-acetylharbazoside nanocomposition were evaluated using dynamic light scattering (DLS) technology to verify its small particle size and low PDI characteristics. This technique determines the hydrated particle size and distribution width based on Brownian motion. Samples were dispersed at 1 mg / mL in purified water, equilibrated at 25°C for 2 minutes, and then measured three times consecutively.

[0109] The dry-basis loading of the lyophilized 8-O-acetylharbazoside nanocomposition was evaluated. The dry-basis loading was calculated as the percentage of the mass of 8-O-acetylharbazoside to the total dry mass of the 8-O-acetylharbazoside-soybean lecithin complex, lactobionic acid and thioglycolic acid-modified chitosan, and sodium tripolyphosphate. The lyophilization protectant was not included in the loading baseline to verify the balance between high drug loading and the formulation. After sample dissolution, the peak area of ​​8-O-acetylharbazoside was quantified using high-performance liquid chromatography (HPLC). Specifically, the sample was accurately weighed, extracted ultrasonically using a methanol-water system, filtered, and then injected. A C18 column was used at a flow rate of 1.0 mL / min. The detection wavelength was determined according to the methodology, and quantification was performed using the external standard method (n=3).

[0110] Ethanol residue was evaluated in the lyophilized powder of 8-O-acetylharbazoside nanocomposition to verify the effectiveness of the de-alcoholization process. Volatile organic solvents were separated by headspace gas chromatography, and ethanol was quantified using a standard curve. Samples were weighed, placed in headspace vials, diluted, sealed, and equilibrated at 80°C for 30 minutes before injection for detection (n=3).

[0111] The moisture content of the lyophilized powder of 8-O-acetylharbazoside nanocomposition was evaluated to verify its low moisture content and storage suitability. The Karl Fischer method was used to determine the moisture content of the samples via the quantitative reaction of iodine with water. After precise weighing, volumetric titration was performed, with three replicates per batch. Samples were rapidly transferred away from moisture and blank correction was performed (n=3). The moisture content (wt%) and mean ± standard deviation were reported and compared with the 5wt% control target.

[0112] The rapid redispersibility of the lyophilized 8-O-acetylharbazoside nanoparticle reconstituted system was evaluated. Particle size changes before and after reconstitution reflected the degree of aggregation / deaggregation, and electrophoretic light scattering was used to assist in determining the recovery of interfacial electrical properties. Purified water was added to the lyophilized powder according to the original solid content, and the particle size was measured after gentle shaking for 1 minute. Zeta potential was measured simultaneously if necessary. Measurements were taken twice (n=3) at 25℃, at 1 minute and 30 minutes after reconstitution.

[0113] The in vitro release behavior of oral or lyophilized reconstituted formulations of 8-O-acetylharbazoside nanocompositions was evaluated to verify the release and absorption windows. The drug release ratio over time was determined in a specified dissolution medium using a paddle method. The drug was first dissolved in pH 1.2 medium for 30 minutes, then transferred to pH 6.8 phosphate buffer for 6 hours. Samples were taken periodically and quantified by HPLC. The experiment was conducted at 37.0 ± 0.5 °C and 50 rpm (n=6). Cumulative release curves were plotted, and the release rates at 2 hours and 6 hours were reported.

[0114] Accelerated storage stability evaluation was conducted on the lyophilized powder of 8-O-acetylharbazoside nanocomposition. Changes in particle size, PDI, water content, and residual ethanol were examined under accelerated conditions to determine the formulation window robustness. Sealed samples were stored at 40℃±2℃ / 75%RH±5%RH for 30 days, and particle size / PDI, water content, and residual ethanol were measured at 0, 15, and 30 days (n=3 batches).

[0115] Figure 1 The overlay images of DLS particle size distributions for Example 1 and Comparative Example 10 show the particle size intensity distribution of the freeze-dried and reconstituted system characterized by dynamic light scattering. The results show that the main peak of Example 1 is concentrated and narrow, with the particle size distribution mainly concentrated in the smaller particle size range. In contrast, the distribution of Comparative Example 10 is significantly wider and shows tailing towards the larger particle size, indicating that the construction of the ion-crosslinked shell is beneficial for suppressing particle aggregation and improving the uniformity of nano-dispersion.

[0116] Figure 2 The particle size recovery curves after rehydration for Examples 1, 7, and 10 are shown. The particle size recovery rate was calculated using dynamic light scattering measurements at different time points after lyophilization and rehydration. The results show that Example 1 can quickly recover to a particle size close to that before lyophilization in the initial stage of rehydration, and the recovery process is stable. In contrast, Comparative Examples 7 and 10 recover more slowly and have lower final recovery rates, indicating that Example 1 has a greater advantage in structural reconstruction and redispersion after lyophilization.

[0117] Figure 3 The time-particle size diagrams for Examples 1, 7, and 10 are shown. The average particle size change at different time points after reconstitution was continuously measured using dynamic light scattering. The results show that the average particle size change after reconstitution in Example 1 is smaller and can quickly stabilize in a range close to the initial level, while the particle size in Comparative Examples 7 and 10 is larger in the early stage and the stabilization rate is slower, indicating that Example 1 has better structure retention and reconstitution stability.

[0118] Figure 4 The FTIR overlays of Examples 1, 8, and 9 show the interfacial chemical characteristics of different shell material systems, which were characterized by Fourier transform infrared spectroscopy. The results show that Example 1 exhibits characteristic changes distinct from single-modification systems in the carbonyl, amide, and sulfur-related absorption regions, indicating that lactobionic acid substitution and thioglycolic acid modification are not simply additive but jointly participate in the construction of the shell interfacial chemical environment.

[0119] Figure 5The in vitro release curves for Examples 1, 8, and 9 were obtained. The cumulative release rate at different time points was determined using a paddle dissolution method combined with high-performance liquid chromatography (HPLC). The results showed that Example 1 maintained a relatively balanced drug release behavior throughout the release process, avoiding both structural instability caused by excessively rapid release and insufficient effective release caused by excessively slow release. This indicates that the dual-modified shell helps to form a release window that balances stability and release efficiency.

[0120] Figure 6 The image shows a scanning electron microscope (SEM) image of the 8-O-acetylharbazoside nanocomposition prepared in Example 1. Figure 6 A low-magnification image shows that the freeze-dried powder has a loose, porous, sponge-like skeletal structure with uniformly distributed pores ranging from 5 to 15 micrometers in size, without large areas of dense areas or cracks. Figure 6 The high-magnification image (b) reveals that the spherical nanoparticles have an average particle size of approximately 150 nm and are uniformly distributed. The particles are connected by a freeze-drying protectant matrix, maintaining a relatively independent spherical profile. This demonstrates that the ion crosslinking method combined with freeze-drying can effectively control the particle size uniformity and core-shell structure integrity of the nanoparticles. At the same time, the mild low-temperature drying conditions avoid irreversible agglomeration between particles, ensuring rapid redispersibility after rehydration.

[0121] Figure 7 Transmission electron microscopy (TEM) images of the 8-O-acetylharbazoside nanocomposite prepared in Example 1. Figure 7 The bright-field plot shows that the nanoparticles are well dispersed with a particle size of 140 to 160 nm, which is in high agreement with the dynamic light scattering measurement results. Figure 7 b. The magnified view clearly shows the core-shell double-layer structure. The core region with high electron density has a diameter of about 120nm to 130nm and is composed of an 8-O-acetylharbazoside-soybean lecithin complex. It is surrounded by a chitosan shell layer modified with lactobionic acid and thioglycolic acid with a thickness of about 10 to 15nm. The core-shell interface is clear and there is no diffusion layer. Figure 7 High-resolution transmission electron microscopy (HRTEM) images show that both the core and shell phases exhibit amorphous and amorphous characteristics, with no lattice fringes observed. Selected area electron diffraction (SED) patterns show diffuse amorphous halos and irregular diffraction spots, demonstrating that the 1:3 mass ratio of 8-O-acetylharbazoside to soybean lecithin composite process used in this embodiment, combined with a 1:1.5 mass ratio of core to shell phase, and cross-linked with sodium tripolyphosphate at 23°C, can form a core-shell nanocomposite with a complete structure and stable interfacial bonding. Furthermore, the freeze-drying protectant effectively inhibits the crystallization and precipitation of the active ingredient, ensuring the stability of the 25wt% drug loading and good bioavailability.

[0122] Figure 8This is a macroscopic photograph of the lyophilized powder of the 8-O-acetylharbazoside nanocomposition prepared in Example 1. The sample is a light yellow, loose powder with a uniform overall distribution, without obvious lumps or color difference areas. The water content is 2.8 wt%, and the residual ethanol content is 0.12 wt%, demonstrating that D-trehalose dihydrate effectively maintains the dispersion state of the nanoparticles as a lyophilization protectant. Furthermore, the process parameters of 7 hours of pre-freezing combined with 24 hours of main drying ensure sufficient drying and good powder flowability, providing a stable material basis for subsequent capsule formation.

[0123] Table 1 Summary of performance of examples and comparative examples

[0124]

[0125] As can be seen from the performance of the examples and comparative examples in Table 1, Examples 1-4 are generally superior to the comparative examples in key stability indicators such as particle size, polydispersity index, reconstitution particle size recovery rate, and 30-day particle size growth rate. This indicates that the synergistic construction of the 8-O-acetylharbazoside-soybean lecithin composite core phase, the lactobionic acid and thioglycolic acid dual-modified shell layer, and the sodium tripolyphosphate ion crosslinking interface plays a decisive role. Among them, Comparative Examples 3 and 10 show higher dry-basis loading or early release, but are accompanied by particle size enlargement, PDI deterioration, and a significant decrease in reconstitution stability, indicating that simply pursuing high loading or fast release will disrupt the overall balance of the core-shell system. Example 2 is biased towards high loading and relatively fast release, Example 4 is biased towards homogeneity and storage stability, and Example 1 is the most balanced among multiple indicators, making it suitable as an anchor sample for subsequent single-factor experiments.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An 8-O-acetylharbazoside nanocomposition, characterized in that, It includes the following components: 8-O-acetylharbazoside-soybean lecithin complex; lactobionic acid and thioglycolic acid-modified chitosan; sodium tripolyphosphate; The 8-O-acetylharbazoside-soybean lecithin complex is the core phase, and the lactobionic acid and thioglycolic acid dual-modified chitosan forms a shell layer coating the core phase with the sodium tripolyphosphate. The ratio of the 8-O-acetylharbazoside-soybean lecithin complex, the lactobionic acid and thioglycolic acid dual-modified chitosan, and the sodium tripolyphosphate by weight is 1:0.5-3.0:0.02-0.

20. The nanocomposition has a particle size of 80-220 nm and a polydispersity index of 0.10-0.30; the lactobionic acid substitution degree of the lactobionic acid and thioglycolic acid dual-modified chitosan is 2-10 mol%, and the thiol content is 30-180 μmol / g; in the nanocomposition, the shell is formed by the lactobionic acid and thioglycolic acid dual-modified chitosan and the sodium tripolyphosphate through ionic crosslinking, and coats the core phase.

2. The 8-O-acetylharbazoside nanocomposition according to claim 1, characterized in that, The lactobionic acid and thioglycolic acid dual-modified chitosan was prepared by the following steps: A1. Disperse 100 parts by weight of lactobionic acid modified chitosan intermediate in 1000-3000 parts by weight of purified water; A2. Adjust the pH of the system to 4.5-6.0 using sodium hydroxide; A3. Add 5-40 parts by weight of mercaptoacetic acid, 5-30 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 3-20 parts by weight of N-hydroxysuccinimide to the system obtained in step A2, and react at 20-30°C under nitrogen protection for 2-10 hours. A4. Dialyze with purified water for 12-48 hours, then vacuum dry at 35-55°C for 8-24 hours to obtain the lactobionic acid and thioglycolic acid dual-modified chitosan. The thiol content of the lactobionic acid and thioglycolic acid dual-modified chitosan is 30-180 μmol / g, and the residual amount of free thioglycolic acid is not higher than 0.5 wt%.

3. The 8-O-acetylharbazoside nanocomposition according to claim 2, characterized in that, The lactobionic acid-modified chitosan intermediate was prepared through the following steps: B1. Add 100 parts by weight of chitosan to 1000-3000 parts by weight of a 0.5-2.0 vol% aqueous solution of acetic acid prepared by acetic acid and purified water, stir and disperse to obtain a chitosan dispersion system; B2. Adjust the pH of the chitosan dispersion system to 4.5-5.5 using sodium hydroxide; B3. Add 10-60 parts by weight of lactobionic acid, 8-40 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 5-25 parts by weight of N-hydroxysuccinimide to the system obtained in step B2, and react at 20-35°C for 4-12 hours. B4. Dialyze with purified water for 12-48 hours, then vacuum dry at 40-60°C for 8-24 hours to obtain the lactobionic acid modified chitosan intermediate, wherein the lactobionic acid degree of substitution of the lactobionic acid modified chitosan intermediate is 2-10 mol.

4. The 8-O-acetylharbazoside nanocomposition according to claim 3, characterized in that, The dialysis described in step B4 uses a dialysis bag with a molecular weight cutoff of 3000-14000 Da, and is performed in purified water for 12-48 hours, with the purified water being replaced every 4-12 hours.

5. The 8-O-acetylharbazoside nanocomposition according to claim 1, characterized in that, The 8-O-acetylharbazoside-soybean lecithin complex was prepared by the following steps: C1. Add 10-50 parts by weight of 8-O-acetylharbazoside and soybean lecithin to 300-3000 parts by weight of ethanol, wherein the mass ratio of 8-O-acetylharbazoside to soybean lecithin is 1:1-1:6; C2. Stir at 40-60℃ for 0.5-4 hours; C3. After removing ethanol under reduced pressure at 35-45℃, dry under vacuum at 25-40℃ for 4-12 hours. C4. The 8-O-acetylharbazoside-soybean lecithin complex is obtained, wherein the residual ethanol content in the complex is not higher than 0.5 wt%, and the loading of 8-O-acetylharbazoside in the complex is 10-50 wt%.

6. A method for preparing the 8-O-acetylharbazoside nanocomposition according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Add 100 parts by weight of lactobionic acid and thioglycolic acid dual-modified chitosan to 1000-5000 parts by weight of 0.2-1.0 vol% acetic acid aqueous solution prepared by acetic acid and purified water to obtain the shell phase of lactobionic acid and thioglycolic acid dual-modified chitosan; S2. The 8-O-acetylharbazoside-soybean lecithin complex is added to the shell phase and dispersed at 3000-12000 r / min for 3-15 min to obtain a dispersion system, wherein the mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex to the lactobionic acid and thioglycolic acid dual-modified chitosan is 1:0.5-3.0; S3. Dissolve sodium tripolyphosphate in purified water to obtain an aqueous solution of sodium tripolyphosphate. Add the aqueous solution of sodium tripolyphosphate dropwise to the dispersion system at a temperature of 20-30℃ and a pH of 4.5-6.5, and continue stirring for 10-60 minutes to carry out ionic cross-linking. The mass ratio of the 8-O-acetylharbazoside-soybean lecithin complex, the lactobionic acid and thioglycolic acid-modified chitosan, and the sodium tripolyphosphate is 1:0.5-3.0:0.02-0.

20. S4. Subsequently, freeze-drying was performed to obtain the 8-O-acetylharbazoside nanocomposition.

7. The preparation method according to claim 6, characterized in that, In step S2, the dispersion speed is 5000-10000 r / min and the dispersion time is 5-10 min; in step S3, the sodium tripolyphosphate aqueous solution is added for 15-40 min, the ion crosslinking temperature is 20-25℃, and the pH value of the ion crosslinking system is 5.0-6.

0.

8. The preparation method according to claim 6, characterized in that, In step S4, based on the total mass of the 8-O-acetylharbazoside-soybean lecithin complex, the lactobionic acid and thioglycolic acid double-modified chitosan, and the sodium tripolyphosphate, a lyophilization protectant of 5-30 wt% is added. The lyophilization protectant is selected from one or both of D-trehalose dihydrate and D-mannitol. The pre-freezing temperature is -40℃ to -20℃, the pre-freezing time is 2-12 h, the main drying pressure is 0.00002-0.00010 MPa, and the main drying time is 12-36 h.

9. The preparation method according to claim 6, characterized in that, The obtained 8-O-acetylharbazoside nanocomposition has an ethanol residue of no more than 0.5 wt% and a lyophilized powder moisture content of no more than 5 wt%.

10. The method according to claim 6, characterized in that, After step S4, a formulation forming step is also included, wherein the obtained 8-O-acetylharbazoside nanocomposition is made into oral formulation raw material powder, granules, capsule contents, tablet granule intermediates, oral suspensions or oral lyophilized reconstituted formulations; when an oral formulation with a unit dose is prepared, the unit dose is 1-100 mg based on 8-O-acetylharbazoside.

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