A liver inflammation regulating alcoholism-protecting pharmaceutical composition through NF-κB pathway

Abstract

The present application relates to the technical field of pharmaceutical preparation, and discloses a kind of quenching medicine composition for alcohol by regulating liver inflammation through NF-κB channel, including core active monomer consisting of quercetin, kaempferol and aloe emodin, HPMCAS-HF type skeleton network polymer, L-arginine, poloxamer 188, and by PEG 400 and deionized water composition transient mass transfer fluid, each raw material is made into amorphous powder by hot melt extrusion reaction through co-rotating twin-screw extruder.The processing temperature is reduced by the aid of transient mass transfer fluid plasticizing, and the degradation of active monomer by heat is avoided;The monomer is stabilized in amorphous form by polymer skeleton, and the local dissolution microenvironment is adjusted by L-arginine, which overcomes the defect of poor water solubility of natural monomer.The present application improves the dissolution and bioavailability of the drug, so that multiple components synergistically inhibit the NF-κB inflammatory pathway, and enhance the effect of quenching liver.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a hepatoprotective drug composition that regulates liver inflammation through the NF-κB pathway. Background Technology

[0002] Alcohol-induced liver damage is often accompanied by a significant inflammatory response, in which activation of the NF-κB signaling pathway plays a crucial role. Currently, some monomeric components extracted from natural plants, such as quercetin, kaempferol, and aloe-emodin, have been shown to intervene in the NF-κB pathway, downregulating the expression of inflammatory factors, and possessing medicinal potential for alcohol detoxification and liver protection.

[0003] However, these natural active monomers generally possess regular crystal structures and high lattice energies, resulting in low solubility in aqueous media. In oral administration, the drug dissolves slowly in gastrointestinal fluids, making effective absorption difficult and limiting overall bioavailability, thus failing to achieve the necessary blood concentrations to exert its therapeutic effect at the target tissue. Traditional pharmaceutical processing methods, such as physical mixing and pulverization or conventional wet granulation, cannot fundamentally alter the inherent thermodynamic properties of the monomers and cannot effectively overcome their poor solubility.

[0004] To improve the dissolution of poorly soluble drugs, existing technologies often employ hot-melt extrusion processes to prepare solid dispersions, aiming to transform the drug into an amorphous form. However, most of these natural monomers possess certain thermosensitive properties, and the high-temperature environment required by conventional hot-melt extrusion processes can easily lead to thermal degradation and inactivation of the active ingredients. Furthermore, when multiple natural active monomers with different molecular structures are co-loaded into the polymer backbone, due to differences in intermolecular compatibility, drug molecules are prone to aggregate and recrystallize during the cooling phase after high-temperature melting or during later storage. This recrystallization phenomenon disrupts the amorphous dispersion state of the composition, causing a further decrease in drug dissolution. Therefore, existing technologies struggle to simultaneously ensure both chemical stability during processing and physical phase stability during product storage when developing such multi-component natural hangover relief and liver protection drugs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a hepatoprotective drug composition that regulates liver inflammation through the NF-κB pathway. This composition solves the problems of poor water solubility and high crystallization tendency of existing natural active monomers in hepatoprotective applications, which result in low bioavailability. Furthermore, conventional physical mixing or crystallization processes cannot effectively overcome the dissolution barriers of poorly soluble drugs, making it difficult to achieve effective blood drug concentrations in target tissues to regulate liver inflammatory pathways.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a hepatoprotective drug composition for relieving hangovers by regulating liver inflammation through the NF-κB pathway, which is produced by hot-melt extrusion reaction of raw materials comprising the following parts by weight using a co-rotating twin-screw extruder: The composition comprises 6.67 to 13.34 parts quercetin, 6.67 to 13.33 parts kaempferol, 6.66 to 13.33 parts aloe-emodin, 40.0 to 60.0 parts HPMCAS-HF polymer, 10.0 to 20.0 parts L-arginine, 3.0 to 8.0 parts poloxamer 188, 2.0 to 5.0 parts PEG 400, and 1.13 to 4.0 parts deionized water. The quercetin, kaempferol, and aloe-emodin together constitute the core active monomers of the composition. The HPMCAS-HF polymer acts as a backbone network polymer and encapsulates the core active monomers during hot melt extrusion to form an amorphous dispersion system. The L-arginine acts as a microenvironment modifier, regulating the microscopic physicochemical environment of the core active monomers within the system. The poloxamer 188 acts as a compatibility wetting agent, promoting the uniform dispersion of the active substances within the backbone network. The PEG 400 and the deionized water form a transient mass transfer fluid, which is continuously injected into the hot melt extrusion mixing section to promote the full mixing and reaction of solid and liquid materials. Finally, the mixture is cooled and solidified to form an amorphous powder composition.

[0007] By adopting the above technical solution, and due to the synergistic combination of multiple components and a specific hot melt extrusion process, the following effects are achieved: Based on existing experimental results, the relevant effects of this application's scheme can be understood from the following aspects: (1) At the pharmacodynamic level, quercetin, kaempferol, and aloe-emodin all showed regulatory effects on NF-κB-related inflammatory responses under relevant experimental conditions, possibly by weakening p65 nuclear translocation and downregulating the expression of some inflammatory factors. The combination of the three monomers in a specific ratio is expected to help enhance the overall anti-inflammatory effect.

[0008] (2) At the phase transformation level, under the combined action of shear force and heat energy of the hot melt extruder, the original lattice structure of the three active monomers is destroyed. The monomer molecules are dispersed in the three-dimensional network framework of the HPMCAS-HF type polymer in a free state or in the form of amorphous particles. The HPMCAS-HF type polymer provides a steric hindrance effect, inhibiting the aggregation and renucleation pathway of the active monomer molecules, and keeping them in a thermodynamic metastable state.

[0009] (3) In terms of process protection, a transient mass transfer fluid consisting of PEG 400 and deionized water is injected into the mixing zone. Water molecules and PEG segments penetrate the gaps between solid powders, producing a plasticizing effect and reducing the glass transition temperature and melt viscosity of the material system. This reaction process keeps the extrusion processing temperature below the thermal degradation temperature of the monomer molecules, avoiding damage to heat-sensitive substances. Subsequently, the moisture is removed in the vacuum devolatilization stage, the system viscosity recovers, and solidification is completed.

[0010] (4) At the microenvironment regulation level, L-arginine possesses basic amino acid properties, forming a locally alkaline microenvironment on the surface of drug particles when the dispersion system comes into contact with the digestive fluid. Since HPMCAS-HF type polymers are pH-dependent, the weak alkalinity of L-arginine promotes the ionization and dissociation of the carboxyl groups on the polymer side chains, accelerating the penetration of liquid into the matrix. At the same time, intermolecular hydrogen bonds are formed between the amino group of L-arginine and the phenolic hydroxyl group of the active monomer, stabilizing the amorphous phase. Poloxamer 188 reduces the surface tension at the interface between the matrix and the liquid phase, promoting the wetting of the matrix interior by water.

[0011] The raw materials are as follows: 10.0 parts quercetin, 10.0 parts kaempferol, 10.0 parts aloe-emodin, 50.0 parts HPMCAS-HF polymer, 15.0 parts L-arginine, 5.0 parts poloxamer 188, 3.5 parts PEG 400, and 2.33 parts deionized water.

[0012] By adopting the above technical solution, the ratio of total drug loading of active monomers to backbone polymer reaches a suitable state within the compatibility threshold, maintaining the system's anti-crystallization ability during long-term storage.

[0013] The PEG 400 and the deionized water are mechanically mixed evenly at an ambient temperature of 20°C to 25°C and then allowed to stand at room temperature to degas and eliminate internal microbubbles, forming a uniform, transparent and colorless transient mass transfer fluid.

[0014] By adopting the above technical solution, the fluid completes homogeneous transformation and eliminates dissolved gases before being injected into the extruder, preventing melt pressure fluctuations caused by the expansion of bubbles when heated in the mixing zone, and ensuring the uniformity of the extruded strip size.

[0015] The quercetin, kaempferol, aloe-emodin, HPMCAS-HF polymer, and L-arginine were pre-screened using an 80-mesh industrial stainless steel sieve; the waxy poloxamer 188 was coarsely screened using a 20-mesh sieve to deagglomerate; all the screened solid materials were mixed and homogenized in a clean room with a relative humidity of no more than 40% to form a physically dry powder.

[0016] By adopting the above technical solution, the particle size distribution range of each raw material is unified, the flowability segregation caused by the difference in powder bulk density is eliminated, and the consistency of the content of each component per unit volume before entering the extruder is ensured.

[0017] The preparation process includes the following steps: The solid materials in the raw materials are sieved according to the pre-set mass proportions and then fed into a three-dimensional motion mixer to obtain a premixed powder; the PEG 400 and deionized water are stirred to dissolve and allowed to stand for degassing before being loaded into a liquid-phase metering pump for later use; the premixed powder is fed into a co-rotating twin-screw extruder at a constant feeding rate and passes through the feeding zone, conveying zone, and initial melting zone in sequence; after entering the mixing zone, the premixed powder is continuously injected into the barrel using the liquid-phase metering pump, and pressure is generated inside the mixing zone through the internal mixing screw element to promote thorough mixing and reaction of the solid and liquid materials; the mixed material then enters the devolatilization zone, and the vacuum pump is turned on to keep the absolute pressure of the devolatilization zone under negative pressure to remove moisture from the system and perform thermal compensation heating to maintain melt fluidity; the devolatilized melt is extruded in strip form through the extrusion zone and die and then quickly enters the traction cold roller system or pressure roller system for cooling and solidification at a constant cooling rate; finally, the solidified material is pulverized and sieved to obtain the amorphous composition powder.

[0018] By adopting the above technical solutions, continuous flow production is achieved, enabling solid mixing to achieve homogeneous dispersion at the molecular level. Delayed injection of liquid additives prevents powder from absorbing moisture and agglomerating in the feeding section; vacuum devolatilization and thermal compensation maintain the rheological properties required for the melt to pass through the forming die; the traction cooling process can quickly fix the motion state of polymer macromolecular chains and maintain the amorphous spatial arrangement of active monomers.

[0019] In the process of preparing the premixed powder, the solid material is fed into the mixing cylinder of the three-dimensional motion mixer and the total loading coefficient of the material is controlled to be between 40% and 50% of the total volume of the mixing cylinder; the operating speed of the three-dimensional motion mixer is set to 15 to 25 revolutions per minute and the mixing time is set to 20 to 30 minutes, and the machine is stopped and left to stand for dust to fall after the mixing process is completed.

[0020] By adopting the above technical solution, sufficient space for material convection and shearing is provided, breaking the electrostatic agglomeration between particles and improving the mixing uniformity of dry-mixed powder.

[0021] During the operation of adding the premixed powder to the co-rotating twin-screw extruder and sequentially passing through the front section, the feeding rate of the premixed powder is controlled between 2.0 and 5.0 kg per hour; the screw speed is set to 200 to 250 rpm; the temperature of the feeding zone is set between 20°C and 30°C; the temperature of the conveying zone is set between 70°C and 90°C; and the temperature of the initial melting zone is set between 115°C and 125°C.

[0022] By adopting the above technical solution and setting a stepped heating curve, the material is prevented from melting too early at the feed end, which could cause the screw to seize up, thus ensuring a smooth transition of the solid particle group to the viscous flow state.

[0023] During the process of the premixed powder entering the mixing zone, the barrel temperature of the mixing zone is set between 135°C and 145°C; the liquid phase metering pump is used to continuously inject the liquid phase additive into the barrel and the system pressure reaches between 1.5 MPa and 2.0 MPa.

[0024] By adopting the above technical solution, under the set temperature range and back pressure, a dispersive mixing force is generated inside the extruder cavity, which promotes the rapid penetration of mass transfer fluid into the polymer crystal region and accelerates the homogeneous fusion reaction of the overall material.

[0025] During the process of the mixed reaction material entering the devolatilization zone, the absolute pressure of the devolatilization zone is controlled between -0.098 MPa and -0.090 MPa for exhaust and dehumidification; the heating compensation temperature of the devolatilization zone is set between 135°C and 140°C.

[0026] By adopting the above technical solution, free moisture is removed under high vacuum, reducing the residual moisture content of the powder product and extending the stability period of the composition.

[0027] In the extrusion and cooling curing operation, the temperature of the extrusion zone and the die is set between 130°C and 135°C; after the melt is extruded, it enters the traction cooling roller system or the pressure roller system with a surface temperature set between 5°C and 10°C; the cooling rate is controlled between 50°C and 80°C per second for cooling curing, and the final cured material is crushed through a 60 to 80 mesh sieve.

[0028] By adopting the above technical solution, the nucleation and growth window of the material transitioning from the glassy state to the crystalline state is cut off at a high cooling rate, forcing the system to enter the glassy transformation process, and finally obtaining a dry powder with high surface area and high solubility.

[0029] This invention provides a hepatoprotective drug composition for relieving hangovers by regulating liver inflammation through the NF-κB pathway. It possesses the following beneficial effects: 1. This invention combines quercetin, kaempferol, and aloe-emodin in specific mass proportions as the core active monomers. These three components work together upstream of the NF-κB signaling pathway, inhibiting the phosphorylation of related kinases and suppressing the expression of pro-inflammatory cytokines. Through the synergistic anti-inflammatory effect among the multiple components, the efficacy of the composition in regulating liver inflammation pathways is improved, thereby enhancing the effects of alcohol detoxification and liver protection.

[0030] 2. This invention utilizes HPMCAS-HF type polymers as a backbone network, disrupting the original lattice structure of the active monomers during hot melt extrusion, allowing them to be stably dispersed in an amorphous state within the polymer matrix. The added L-arginine creates a locally alkaline microenvironment upon contact with body fluids, promoting the dissociation of the pH-dependent dissolution backbone polymer. Combined with the wetting effect of poloxamer 188, this overcomes the poor water solubility of natural active monomers, improving drug dissolution and bioavailability.

[0031] 3. In the hot melt extrusion compounding stage, this invention continuously injects a mass transfer fluid composed of PEG 400 and deionized water. This fluid's plasticizing effect lowers the glass transition temperature and melt viscosity of the material system, keeping the processing temperature below the thermal degradation temperature of the monomer molecules and preventing heat-sensitive substances from being damaged during processing. Combined with vacuum devolatilization to remove moisture and rapid cooling after molding, the movement of the polymer macromolecular chains is stabilized, ensuring the physical stability of the amorphous powder composition and the continuity of the preparation process. Attached Figure Description

[0032] Figure 1 This is a diagram showing the three-dimensional structure of the complexes formed by each monomer of the present invention and its corresponding target protein, as well as the interaction diagram of the core amino acid residues. Figure 2 The Western Blot gel electrophoresis results and relative protein content line graph of the TNF-α target protein of the present invention during the heating process are shown. Figure 3 The Western Blot gel electrophoresis results and relative protein content line graph of the IL-1β target protein of the present invention during the heating process; Figure 4 The Western Blot gel electrophoresis results and relative protein content line graph of the IL-6 target protein of the present invention during the heating process; Figure 5 The image shows the results of immunofluorescence staining and nuclear translocation quantification of p65 protein in each group of cells according to the present invention. Figure 6 This is a Western blotting gel electrophoresis image of the total cellular protein extract of the present invention. Figure 7 This is a bar chart showing the relative mRNA expression levels of the genes in this invention. Figure 8Figure 1 shows the physicochemical index test results of the lipopolysaccharide-induced acute liver injury model in mice of the present invention. Figure 2 shows the test results of the wet / dry weight ratio (W / D) of liver tissue in each group of mice, Figure 3 shows the test results of the TNF-α concentration in the liver tissue homogenate of each group of mice, Figure 4 shows the test results of the IL-1β concentration in the liver tissue homogenate of each group of mice, and Figure 5 shows the test results of the IL-6 concentration in the liver tissue homogenate of each group of mice. Figure 9 The figures show the pharmacokinetic (plasma concentration-time) curves of the monomeric compounds of the present invention in rats, where Figures (a), (b), and (c) represent the test results of three different monomeric components, respectively. Figure 10 Figure 1 shows the physical stability test results of the solid dispersion of the present invention under accelerated testing conditions. Figure 2(a) is a bar chart of the relative crystallinity change of each monomer component in the solid dispersion powder, and Figure 3(b) is a line graph of the cumulative dissolution rate change of each monomer component in the solid dispersion powder over 45 minutes. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0034] Preparation Examples 1-2: Preparation Example 1: This preparation example provides a method for preparing a specific transient subcritical mass transfer fluid, including the following steps: (1) Weigh out room temperature liquid polyethylene glycol 400 (PEG 400) and deionized water according to the mass fractions preset in the formula.

[0035] (2) Add the weighed PEG 400 to a stainless steel mixing tank equipped with a constant temperature jacket and a mechanical stirring device.

[0036] (3) Under the condition of an ambient temperature of 20℃-25℃, slowly and evenly pour deionized water into the solution tank.

[0037] (4) Turn on the mechanical stirrer and set the stirring speed to 100-150 rpm. Stir continuously for 10-15 minutes until there are no vortex dead corners on the liquid surface and the mixture presents a uniform, transparent, colorless phase.

[0038] (5) Stop stirring and let it stand at room temperature for 5-10 minutes to degas and eliminate tiny air bubbles inside.

[0039] (6) Transfer the uniformly mixed transient mass transfer fluid into the sealed high-pressure liquid metering pump storage tank located on the side of the hot melt extruder, and seal it at room temperature for later use.

[0040] Preparation Example 2: This preparation example provides a method for preparing homogeneous premixed powder of solid materials, including the following steps: (1) In a cleanroom where the relative humidity is controlled at ≤40% and the ambient temperature is 20℃-25℃, weigh the core active monomers (quercetin, kaempferol, aloe-emodin), the backbone network polymer (HPMCAS-HF type), the microenvironment regulator (L-arginine) and the compatibility wetting agent (poloxam 188) according to the mass fractions preset in the formula.

[0041] (2) The core active monomer, HPMCAS-HF type polymer and L-arginine are pre-screened through an 80-mesh industrial stainless steel screen; for the waxy poloxamer 188, a 20-mesh screen is used for coarse screening to deagglomerate.

[0042] (3) The sieved solid materials are sequentially fed into the mixing drum of the three-dimensional motion mixer, and the total loading coefficient of the materials is controlled between 40% and 50% of the total volume of the drum.

[0043] (4) Seal the mixer drum and turn on the grounding and static electricity removal device built into the equipment. Set the operating speed of the mixer to 15-25 rpm and the mixing time to 20-30 minutes.

[0044] (5) After the mixing process is completed, stop the machine and let it stand for 3 minutes to allow the dust to fall back down. Then, unload the fully homogenized physical dry-mixed powder.

[0045] (6) The obtained premixed powder is put into an antistatic storage bin lined with a moisture-proof aluminum-plastic composite bag, and sealed to isolate the ambient moisture. It is then transferred to the constant weight loss feeder of the hot melt extruder for later use.

[0046] Examples 1-3: Example 1: This example provides a hepatoprotective drug composition for relieving hangovers by regulating liver inflammation through the NF-κB pathway, comprising the following steps: (1) Weighing raw materials: Weigh 6.67 parts of quercetin, 6.67 parts of kaempferol, 6.66 parts of aloe-emodin, 40.0 parts of HPMCAS-HF, 10.0 parts of L-arginine, 3.0 parts of poloxamer 188, 2.0 parts of PEG 400, and 1.13 parts of deionized water by mass.

[0047] (2) Material pretreatment: In an environment with relative humidity ≤40% and temperature 22℃, the above three monomers, HPMCAS-HF and L-arginine are passed through an 80-mesh sieve, and poloxamer 188 is passed through a 20-mesh coarse sieve; they are put into a three-dimensional mixer and mixed at 15 rpm for 20 minutes to obtain premixed powder; at the same time, PEG 400 and deionized water are stirred to dissolve each other, and after standing to degas, they are loaded into a liquid phase metering pump.

[0048] (3) Solid feeding and initial melting zone operation: The premixed powder is fed into the co-rotating twin-screw extruder (L / D=40) at a feeding rate of 2.0 kg / h. The screw speed is set to 200 rpm. The extruder front section temperature is set as follows: 20℃ in the feeding zone, 70℃ in the conveying zone, and 115℃ in the initial melting zone.

[0049] (4) Liquid-assisted mixing section operation: The material enters the mixing zone and the barrel temperature of the zone is set to 135°C. The liquid phase additive is continuously injected into the barrel using a metering pump. The pressure in this section is made to reach 1.5MPa through the configuration of the threaded element, so that the solid and liquid materials are fully mixed and reacted.

[0050] (5) Vacuum devolatilization section operation: When the material enters the devolatilization zone, the vacuum pump is turned on to reduce the absolute pressure of the zone to -0.090MPa and remove the moisture in the system. At the same time, the barrel heating temperature of the zone is set to 135℃ to compensate for the latent heat of devolatilization and maintain the melt fluidity of the material.

[0051] (6) Extrusion molding and cooling curing: The devolatilized melt is extruded in strip form through a 130°C extrusion zone and a 130°C die, and enters a traction cooling roller system with a surface temperature of 10°C within 1.0 second. The cooling rate is controlled at 50°C / s for cooling curing. Finally, the cured material is crushed and passed through a 60-mesh sieve to obtain an amorphous composition powder.

[0052] Example 2: This example provides a hepatoprotective drug composition for relieving hangovers by regulating liver inflammation through the NF-κB pathway, comprising the following steps: (1) Weigh the raw materials: weigh 10.0 parts of quercetin, 10.0 parts of kaempferol, 10.0 parts of aloe-emodin, 50.0 parts of HPMCAS-HF, 15.0 parts of L-arginine, 5.0 parts of poloxamer 188, 3.5 parts of PEG 400, and 2.33 parts of deionized water by mass.

[0053] (2) Material pretreatment: In an environment with relative humidity ≤40% and temperature 22℃, monomers, HPMCAS-HF and L-arginine are passed through an 80-mesh sieve, and poloxamer 188 is passed through a 20-mesh sieve; they are put into a three-dimensional mixer and mixed at 20 rpm for 25 minutes to obtain premixed powder; PEG 400 and deionized water are mixed for later use.

[0054] (3) Solid feeding and initial melting zone operation: The premixed powder is fed into the co-rotating twin-screw extruder at a feeding rate of 3.5 kg / h. The screw speed is set to 225 rpm. The extruder front section temperature is set as follows: 25℃ in the feeding zone, 80℃ in the conveying zone, and 120℃ in the initial melting zone.

[0055] (4) Liquid-assisted mixing section operation: The cylinder temperature of the mixing zone is set to 140℃. Liquid phase additives are pumped in, and the pressure inside the section reaches 1.75MPa through the internal mixing thread element.

[0056] (5) Vacuum devolatilization section operation: The material enters the devolatilization zone, and the absolute pressure is controlled at -0.095MPa for exhaust and dehumidification. The heating temperature of this section is set to 138℃.

[0057] (6) Extrusion molding and cooling solidification: The melt is pushed to the 133°C extrusion zone and 133°C die for extrusion, and enters the traction cooling roller with a surface temperature of 8°C. The cooling rate is controlled at 65°C / s for cooling solidification. After pulverization, it is passed through an 80-mesh sieve to obtain the drug composition powder.

[0058] Example 3: This example provides a hepatoprotective drug composition for relieving hangovers by regulating liver inflammation through the NF-κB pathway, comprising the following steps: (1) Weighing raw materials: Weigh 13.34 parts of quercetin, 13.33 parts of kaempferol, 13.33 parts of aloe-emodin, 60.0 parts of HPMCAS-HF, 20.0 parts of L-arginine, 8.0 parts of poloxamer 188, 5.0 parts of PEG 400, and 4.0 parts of deionized water by mass.

[0059] (2) Material pretreatment: In an environment with relative humidity ≤40% and temperature 25℃, monomers, HPMCAS-HF and L-arginine are passed through an 80-mesh sieve, and poloxamer 188 is passed through a 20-mesh sieve; they are put into a mixer and mixed at 25 rpm for 30 minutes to obtain premixed powder; the corresponding PEG / water fluid is prepared for later use.

[0060] (3) Solid feeding and initial melting zone operation: The premixed powder is fed into the twin-screw extruder at a rate of 5.0 kg / h. The screw speed is set to 250 rpm. The front-end temperatures are set as follows: 30°C in the feeding zone, 90°C in the conveying zone, and 125°C in the initial melting zone.

[0061] (4) Liquid-assisted mixing section operation: The cylinder temperature in the mixing zone is set to 145℃. Liquid phase additives are pumped in, and the system pressure reaches 2.0MPa.

[0062] (5) Vacuum devolatilization section operation: Enter the devolatilization zone and the absolute pressure drops to -0.098MPa to remove moisture. The system thermal compensation temperature is set to 140℃.

[0063] (6) Extrusion molding and cooling solidification: The melt is extruded through a 135°C extrusion zone and a 135°C die, and enters a pressure roller system with a surface temperature of 5°C. The cooling rate is controlled at 80°C / s for cooling solidification, and the mixture is pulverized and passed through an 80-mesh sieve to obtain the drug composition powder.

[0064] Comparative Examples 1-5: Comparative Example 1: Compared with Example 2, the difference is that L-arginine is not added to the raw material formula, while the other ratios and preparation steps are the same.

[0065] Comparative Example 2: Compared with Example 2, the difference is that: no transient mass transfer fluid is prepared (PEG 400 and deionized water are not added to the raw materials), no liquid injection operation is performed during the extrusion process, and pure dry mixing extrusion is used. The remaining proportions and preparation steps are the same.

[0066] Comparative Example 3: Compared with Example 2, the difference is that the vacuum pump is not turned on for devolatilization in the extrusion process, and the material is not rapidly cooled in the cold roller system after being extruded from the die head. Instead, it is naturally cooled and solidified at room temperature (25°C). The other proportions and preparation steps are the same.

[0067] Comparative Example 4: Compared with Example 2, the difference is that the preparation process does not use a twin-screw hot melt extruder. Instead, all solid and liquid raw materials in the formula are directly mixed at room temperature using a conventional high-shear wet granulation process, followed by drying and pulverization. The proportions of the remaining raw materials are the same.

[0068] Comparative Example 5: Compared with Example 2, the difference is that only 30.0 parts of quercetin monomer are used as the core active monomer component, and kaempferol and aloe-emodin are not added. The other proportions and preparation steps are the same.

[0069] Test Examples 1-6: Test Example 1: Molecular docking test of the affinity between the active ingredient and the target protein This test case employs molecular docking computational techniques to evaluate the physicochemical feasibility of binding three core monomers in the drug composition to key pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The specific experimental steps are as follows: 1. Retrieve and download the three-dimensional crystal structure files of the target proteins TNF-α, IL-1β, and IL-6 from the RCSB PDB database. Use appropriate software to preprocess the protein structures, removing original ligand molecules, water molecules, and non-standard residues.

[0070] 2. The two-dimensional molecular structures of quercetin, kaempferol and aloe-emodin were obtained from the PubChem database, converted into three-dimensional structural formats, and then subjected to structural minimization and energy optimization using the MM2 molecular force field.

[0071] 3. Import the pretreated protein and ligand molecules into AutoDockTools software. Add polar hydrogen atoms to the protein, calculate the Gasteiger charge, synthesize nonpolar hydrogen atoms, and save both the receptor and ligand in PDBQT format.

[0072] 4. Based on the reported amino acid residue distribution of the target protein's active site or allosteric regulatory region, set the GridBox grid parameters to ensure that the docking box completely covers the target binding pocket.

[0073] 5. Call the AutoDock Vina program to perform semi-flexible docking calculations, set the global search parameter exhaustiveness to the default value, and record the binding energy data.

[0074] 6. Extract the docking conformation with the lowest binding energy, and use PyMOL and Discovery Studio software to extract and visualize the three-dimensional interaction mode of the receptor-ligand complex.

[0075] The experimentally measured binding energies are shown in the table below: Table 1. Binding energy test data of core active monomers and inflammatory cytokine target proteins

[0076] Appendix Figure 1 The diagram shows the three-dimensional structure of the complexes formed by each monomer and its corresponding target protein, as well as the interaction diagram of the core amino acid residues. (Attached) Figure 1 In the text, "Quercetin" represents quercetin, "Aloe-emodin" represents aloe-emodin, and "Kaempferol" represents kaempferol; "act on" indicates that it acts on; the combination of uppercase letters and numbers represents specific amino acid residues on the target protein; and the dashed lines between the structures represent the intermolecular hydrogen bonds formed.

[0077] According to the data in Table 1, the binding energies of the three core monomers to the inflammatory factors TNF-α, IL-1β, and IL-6 all ranged from -6.65 kcal / mol to -9.14 kcal / mol. The binding energies below -5.0 kcal / mol indicate that the ligands and receptor proteins possess a tendency to form stable, non-covalent, self-heating mechanical bonds. The molecular backbones of quercetin, kaempferol, and aloe-emodin contain free phenolic hydroxyl groups and conjugated fused ring systems. These structures can act as hydrogen bond donors and acceptors, forming multiple hydrogen bond cross-links with polar amino acid residues within the target protein binding pocket. Simultaneously, the aromatic ring system of the monomers can undergo π-π stacking and hydrophobic interactions with residues within the hydrophobic cavity of the protein.

[0078] Combined with appendix Figure 1 The specific modes of action reveal that all three core monomers bind to key regions of inflammatory factors. Quercetin forms a stable interaction with Q102 and S99 residues of the TNF protein. When binding to IL-1β, it resides in a pocket formed by residues such as K63, E64, and G61 through hydrogen bonds and hydrophobic interactions. When acting on IL-6, it binds to residues such as P66, L65, and M58. Aloe-emodin and kaempferol also form dense hydrogen bond networks at the active sites of their respective target proteins. For example, aloe-emodin binds to T44 and L10 residues of IL-6, while kaempferol binds to residues such as Q102 and P100 of TNF. This multidimensional physical binding occupies the active sites of inflammatory factors, interfering with their binding interface with downstream receptors. The above molecular docking results indicate that the three core monomers have good binding energies with TNF-α, IL-1β, and IL-6, suggesting a potential interaction tendency with related inflammatory factors and providing a reference for subsequent mechanistic studies.

[0079] Test Example 2: Cellular Thermal Transfer Analysis (CETSA) of Target Proteins This test case uses cell thermal transfer analysis to evaluate the changes in thermodynamic stability of drug monomers after binding with inflammatory cytokine target proteins under different temperature gradients, verifying the physical binding of the two in complex biological systems. The specific experimental steps are as follows: 1. RAW 264.7 macrophages were cultured. Cells were collected when confluence reached 80%, and lysed on ice for 30 minutes using non-denaturing lysis buffer. The cells were then centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was extracted to obtain the total protein sample. Protein concentration was determined using a BCA assay kit and the concentrations were balanced. Simultaneously, purified solutions of recombinant human TNF-α, IL-1β, and IL-6 at the required concentrations were prepared.

[0080] 2. The RAW 264.7 cell protein extract and recombinant human protein solution were divided into a control group and a treatment group, respectively. An equal volume of solvent was added to the control group, while solutions of quercetin, kaempferol, and aloe-emodin at a final concentration of 50 μM were added to the treatment groups, respectively. Each group of samples was incubated at room temperature in the dark for 45 minutes to allow for sufficient contact and reaction between the drug molecules and the target proteins in the mixture.

[0081] 3. Divide each group of protein samples after incubation into 5 aliquots and transfer them into PCR tubes. Heat-treat the samples using a gradient PCR instrument, setting the temperature gradient to 40℃, 50℃, 60℃, 70℃, and 80℃, with a constant heating time of 3 minutes at each temperature. Immediately after heating, transfer the PCR tubes to room temperature and cool for 3 minutes.

[0082] 4. Centrifuge the samples treated at different temperatures at 20000g for 20 minutes at 4℃ to separate the heat-denatured and aggregated precipitated proteins from the soluble proteins that retain their native conformation.

[0083] 5. Aspirate the supernatant after centrifugation, add an equal volume of SDS-PAGE gel loading buffer, and boil at 95°C for 5 minutes to denature the sample. Separate the processed sample by polyacrylamide gel electrophoresis, and then transfer it onto a PVDF membrane.

[0084] 6. Intervene the PVDF membrane with 5% skim milk for 1 hour. Add specific anti-TNF-α, anti-IL-1β, and anti-IL-6 primary antibodies respectively, and incubate overnight at 4°C. After washing the membrane, add HRP-labeled secondary antibody and incubate at room temperature for 1 hour. Develop the membrane using ECL chemiluminescence solution, acquire images using a gel imaging system, calculate the relative residual amount of soluble target protein at each temperature through grayscale analysis, and calculate the half-maximum denaturation temperature (Tm value) of the target protein by fitting the data.

[0085] The target protein half-denaturation temperature (Tm) data obtained in the experiment are shown in the table below: Table 2. Half-maximum denaturation temperature data of target proteins before and after drug monomer intervention

[0086] Appendix Figure 2 Appendix Figure 3 and attached Figure 4 The Western blotting gel electrophoresis results of TNF-α, IL-1β, and IL-6 target proteins during the heating process described above, along with corresponding line graphs showing their relative protein content, are presented respectively. The English text and labels in the figures are explained below: “RAW 264.7” represents the RAW 264.7 macrophage lysis buffer experimental system; “Human Recombinant Protein” represents the human recombinant protein experimental system; “Temperature (°C)” and the black wedge-shaped gradient bar above it represent the increase in heat treatment temperature gradient (from 40°C to 80°C); “Ctrl” represents the drug-free control group; “QR” represents the quercetin-treated group; “AE” represents the aloe-emodin-treated group; “Kae” represents the kaempferol-treated group; “kDa” is kilodaltons, representing the molecular weight of each target protein; the vertical axis of the line graph, “Relative protein content of…”, represents the relative content of the corresponding target protein in a specific system.

[0087] According to Table 2 and appendix Figure 2 To be continued Figure 4 The data showed that in the control group without drug intervention, whether in the complex biological matrix system of RAW264.7 cell lysate or in the purified human recombinant protein system, as the heating temperature increased from 40℃ to 80℃, the electrophoretic bands of TNF-α, IL-1β and IL-6 proteins gradually became lighter, and their half denaturation temperatures (Tm values) ranged from 48.7℃ to 52.4℃.

[0088] After intervention with quercetin, kaempferol, or aloe-emodin, the protein degradation curves in the line graphs showed an overall shift towards higher temperatures. The Tm values ​​of the three target proteins increased in both experimental systems, with the increase concentrated between 6.2℃ and 10.1℃. When proteins are heated, their higher-order structures unfold and aggregate, while the binding of ligand molecules to the target proteins can reduce the conformational entropy of the protein system and improve its resistance to thermal denaturation.

[0089] According to Table 2 and appendix Figure 2 To be continued Figure 4 The results showed that the Tm values ​​of the relevant target proteins were all increased after treatment with the three monomers compared with the control group, suggesting that they may interact with TNF-α, IL-1β, and IL-6 in the experimental system and may improve the thermal stability of the relevant proteins. This result supports the binding of the three monomers to inflammation-related targets. Combined with subsequent pharmacokinetic and dissolution results, it can be inferred that increased release and in vivo exposure levels of the active ingredients may contribute to the exertion of related pharmacological effects.

[0090] Test Example 3: Pathway Regulation Mechanism Testing in an In Vitro Inflammatory Cell Model This test case used a lipopolysaccharide-induced RAW 264.7 macrophage inflammation model to evaluate the regulatory effects of monomeric compounds on the NF-κB inflammatory signaling pathway and their influence on the transcriptional expression of downstream inflammatory factors at the cellular level. The specific experimental steps are as follows: 1. RAW 264.7 cells in logarithmic growth phase were seeded in DMEM medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator. When cell confluence reached approximately 70%, cells were collected and seeded into 6-well plates and confocal culture dishes.

[0091] 2. The inoculated cells were divided into 5 groups: a control group without intervention, a model group treated with 1 μg / mL lipopolysaccharide, and treatment groups treated with 1 μg / mL lipopolysaccharide along with 50 μM aloe-emodin, kaempferol, and quercetin, respectively. Cells in each group were cultured for another 24 hours.

[0092] 3. Aspirate the culture medium from the confocal culture dish, wash the cells with phosphate-buffered saline (PBFS), and fix with 4% paraformaldehyde at room temperature for 20 minutes. After washing, treat with 0.5% Triton X-100 permeabilization buffer for 15 minutes, and then intervene with 5% bovine serum albumin for 1 hour. Add anti-p65 primary antibody and incubate overnight at 4°C; wash and add FITC-labeled secondary antibody and incubate in the dark for 1 hour. Add DAPI staining solution for nuclear staining, mount the slides, and observe the subcellular localization of p65 protein under a laser confocal microscope.

[0093] 4. Collect cells from each group in 6-well plates, add RIPA lysis buffer containing protease and phosphatase inhibitors to extract total protein. Centrifuge and collect the supernatant, then quantify using the BCA method. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. Intervention was performed using 5% skim milk, with the addition of anti-p-p65, anti-p65, anti-IκB-α, and anti-GAPDH primary antibodies, respectively, and incubated overnight at 4°C. After washing, HRP-labeled secondary antibody was added and incubated at room temperature. Protein expression levels were detected using chemiluminescence immunoassay.

[0094] 5. Total RNA was extracted from cells in each well of a 6-well plate using a total RNA extraction reagent. After determining the concentration and purity, cDNA was synthesized via reverse transcription. Using the cDNA as a template, specific primers and fluorescent dyes targeting TNF-α, IL-1β, iNOS, IL-6, and the internal control GAPDH were added, and amplification was performed on a real-time quantitative PCR instrument. Ct values ​​were read, and the results were analyzed using a 2... (-ΔΔCt) The relative transcriptional expression levels of each target gene were calculated.

[0095] The relative expression levels of inflammatory factors and synthase mRNA obtained from the test are shown in the table below: Table 3. Relative mRNA expression data of inflammation-related genes in RAW 264.7 cells

[0096] Appendix Figure 5The results of immunofluorescence staining and nuclear translocation quantification of p65 protein in each group of cells are presented; (Attached) Figure 6 Western blotting gel electrophoresis bands of total cellular protein extract are shown; (attached) Figure 7 The bar chart shows the relative mRNA expression levels of the target gene. The English text and labels in the chart are explained below: "Control" represents the control group; "LPS" represents the lipopolysaccharide model group; "LPS+AE" represents the lipopolysaccharide and aloe-emodin co-treatment group; "LPS+Kae" represents the lipopolysaccharide and kaempferol co-treatment group; "LPS+QR" represents the lipopolysaccharide and quercetin co-treatment group; "P65" and "p65" represent the nuclear transcription factor p65; "DAPI" is the nuclear fluorescent dye; "Merge" is the merged fluorescence channel image; "Relative fluorescence intensity of p65 in RAW264.7" indicates RAW 264.7 Relative fluorescence intensity of p65 in cells; "p-p65" indicates phosphorylated p65 protein; "IκB-α" indicates nuclear transcription factor repressor protein α; "GAPDH" is the internal reference protein; "kD" is kilodaltons, representing the molecular weight of the protein; "TNF-α", "IL-1β" and "IL-6" represent pro-inflammatory cytokines; "iNOS" represents inducible nitric oxide synthase; "Relative mRNA levels" indicates relative mRNA expression levels; the asterisk "*" above the bar indicates that the difference between groups is statistically significant.

[0097] According to the data in Table 3, the basal mRNA expression levels of TNF-α, IL-1β, iNOS, and IL-6 were extremely low in the control group without lipopolysaccharide stimulation. After lipopolysaccharide stimulation, the transcriptional levels of TNF-α, IL-1β, and iNOS were significantly upregulated in the model group. After intervention with aloe-emodin, kaempferol, and quercetin, the abnormal transcription of TNF-α, IL-1β, and iNOS was inhibited, and their expression levels decreased. Among them, the monomers showed a different regulatory trend on IL-6 than other factors, and its actual transcriptional level increased compensatorily with drug administration.

[0098] Combined with appendix Figure 5 Immunofluorescence microscopy revealed that lipopolysaccharide stimulation induced the aggregation and translocation of the green fluorescent p65 protein into the blue fluorescent nucleus. However, in the groups treated with the three monomeric compounds, the green fluorescence signal in the nucleus was weakened, indicating that p65 was retained in the cytoplasm. Simultaneously, [the following text appears to be incomplete and requires further context: "attachment..."] Figure 6Western blot results showed that the grayscale of the phosphorylated p65 (p-p65) protein band in the treatment group was lighter than that in the model group, and the degradation of the upstream repressor protein IκB-α was alleviated. NF-κB (p65) is a key transcription factor regulating the inflammatory response. Lipopolysaccharide stimulation leads to the degradation of IκB-α and promotes the phosphorylation of p65 into the nucleus, thereby initiating the transcription of multiple downstream inflammatory genes.

[0099] The aforementioned changes in expression and distribution at the cellular level indicate that the core monomer in the composition can penetrate into macrophages and inhibit the overactivation of the NF-κB signaling pathway by suppressing IκB-α degradation and p65 nuclear translocation. Combined with the direct target binding results of the aforementioned test examples, this further suggests the pharmacological mechanism by which the composition of this invention intervenes in free pro-inflammatory proteins in the tissue fluid environment and inhibits the transcription of inflammatory genes within cells.

[0100] Test Example 4: In vivo evaluation test of anti-inflammatory drug efficacy This test case used a lipopolysaccharide-induced mouse alcoholic liver injury model to evaluate the efficacy of the composition in treating acute inflammatory responses and its tissue protective effect at the in vivo animal level. The specific experimental steps are as follows: 1. Healthy SPF-grade male C57BL / 6 mice were selected and acclimatized for 7 days in a standard animal facility. The mice were randomly divided into 5 groups of 10 mice each: a control group, a model group, and groups treated with aloe-emodin, kaempferol, and quercetin.

[0101] 2. The solid dispersion powder containing each monomer component (preferably the pharmaceutical composition powder prepared in Example 2) was prepared into a suspension using sterile physiological saline. Mice in the treatment group were administered the drug by gavage at a dose of 50 mg / kg for 7 consecutive days, once a day. Mice in the control group and model group were administered an equal volume of sterile physiological saline by gavage daily.

[0102] 3. Acute liver injury model was established 1 hour after the last gavage administration on day 7. Except for the control group, mice in the other groups were administered 50% ethanol solution (5 g / kg) by gavage, followed by intraperitoneal injection of lipopolysaccharide solution (5 mg / kg) 2 hours later. Mice in the control group were administered sterile saline by gavage and intraperitoneal injection at the same time points.

[0103] 4. Twenty-four hours after modeling, the mice were weighed and their weight changes were recorded. Blood samples were collected by enucleation, and after standing at room temperature, the serum was separated by centrifugation at 3000 rpm for 10 minutes and stored at -80℃ for later use. The mice were then euthanized by cervical dislocation, the abdominal cavity was opened, and the liver tissue was completely separated and removed. A portion of the liver tissue was weighed and placed in pre-cooled sterile PBS buffer to prepare a liver tissue homogenate, which was then centrifuged and the supernatant was collected.

[0104] 5. The right liver lobe tissue was harvested, weighed, and recorded as wet weight (W). It was then placed in an 80℃ constant temperature drying oven for 48 hours until constant weight, weighed, and recorded as dry weight (D). The wet / dry weight ratio of the liver tissue (W / D) was calculated. The left liver lobe tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with H&E, and the pathological morphological changes of the liver tissue were observed under an optical microscope, and liver injury was scored.

[0105] 6. Take out the frozen serum and liver tissue homogenate samples, and determine the concentration levels of TNF-α, IL-1β and IL-6 in the samples according to the operating instructions of the ELISA test kit.

[0106] The data on liver tissue edema and the concentration of inflammatory factors in liver tissue homogenate obtained in the experiment are shown in the table below: Table 4. Test data of relevant physicochemical indicators in mouse acute liver injury model

[0107] According to Table 4 and Figure 8 ( Figure 8 The data (showing the trends in W / D ratio and inflammatory factor concentrations) indicate that the wet / dry weight ratio of the liver in the control group mice was at a baseline level, and the concentration of inflammatory factors in the liver tissue homogenate was low. After stimulation with alcohol combined with lipopolysaccharide, the wet / dry weight ratio of the liver tissue in the model group mice increased to 7.42 (e.g., ...). Figure 8 As shown in sub-Figure a), the concentrations of TNF-α, IL-1β, and IL-6 in liver homogenate reached 876.51 pg / mL, 412.38 pg / mL, and 654.72 pg / mL, respectively (corresponding to...). Figure 8 (Sub-figures b, c, and d) show significantly elevated levels of inflammatory factors. After gavage administration, liver swelling and edema in mice treated with aloe-emodin, kaempferol, and quercetin were alleviated, and the W / D ratio returned to the range of 4.87 to 5.23; the concentrations of the three inflammatory factors decreased by 50% to 65%.

[0108] Aloe-emodin, kaempferol, and quercetin are poorly soluble compounds. This formulation uses a hot-melt extrusion process to prepare them into solid dispersions, allowing the drug molecules to disperse in an amorphous state within the carrier. This improves the drug's dissolution rate and apparent solubility, ensuring that it enters the bloodstream and accumulates in damaged liver tissue after gavage. Animal experiments showed that the formulation of this invention can reduce the levels of inflammatory factors in the liver tissue of model mice and alleviate liver edema, suggesting a certain protective effect against alcohol / lipopolysaccharide-induced acute liver injury. Combined with the aforementioned in vitro experimental results, it is speculated that this effect may be related to improving the in vivo exposure of the active ingredients and regulating NF-κB-related inflammatory responses.

[0109] Test Example 5: In vivo pharmacokinetic test This test case used SD rats to determine and compare the in vivo pharmacokinetic parameters of the monomer mixture raw materials and the solid dispersion of the present invention, and to evaluate the changes in the oral bioavailability of the administered composition. The specific experimental steps are as follows: 1. Select healthy male SD rats weighing 200 to 220g. Fast them for 12 hours before the experiment, during which time they can drink water freely. Randomly divide the rats into two groups of 6 each: a physical mixture group and a solid dispersion group.

[0110] 2. Take equal proportions of the physical mixture powder of the monomeric compound and the solid dispersion powder prepared by the aforementioned process (taken from Example 2), and prepare drug suspensions using 0.5% sodium carboxymethyl cellulose aqueous solution.

[0111] 3. Both groups of rats were administered the drug via a single gavage at a total dose of 100 mg / kg.

[0112] 4. At 0 hours before and 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after gavage administration, approximately 0.3 mL of blood was collected through the orbital venous plexus and collected in anticoagulant centrifuge tubes containing heparin sodium.

[0113] 5. Collect blood samples and centrifuge at 4000 rpm for 10 minutes at 4℃ to separate plasma. Take 50 μL of plasma sample, add 200 μL of acetonitrile containing internal standard to precipitate protein, vortex mix, centrifuge at 12000 rpm for 10 minutes, and collect the supernatant.

[0114] 6. The concentrations of aloe-emodin, kaempferol, and quercetin in plasma samples were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Blood drug concentration data at different time points were recorded, and the peak concentration (Cmax) and area under the curve (AUC0-t) of each compound were calculated using a non-compartmental model.

[0115] Table 5. Data on core pharmacokinetic parameters in rats after oral administration of different formulations

[0116] According to Table 5 and Figure 9 ( Figure 9 This is a pharmacokinetic plasma concentration-time curve; Figure 9 The data includes three vertically arranged subplots (the horizontal axis represents the sampling time point, and the vertical axis represents the plasma drug concentration). In the physical mixture group, the peak plasma drug concentrations and the area under the curve for the three compounds are at a low level. Figure 9The dashed line represents data for physical mixtures, indicating low blood drug concentrations. Specifically, the AUC0-t values ​​for aloe-emodin, kaempferol, and quercetin were 214.62 ng·h / mL, 156.19 ng·h / mL, and 289.41 ng·h / mL, respectively.

[0117] After solid dispersion processing, the Cmax and AUC0-t values ​​of the drug-treated group increased, and the AUC0-t of quercetin in the solid dispersion group increased to 1436.52 ng·h / mL. Figure 9 The solid line represents the experimental data for the solid dispersion, and subplots a, b, and c represent the results for the three monomer components, respectively. Compared to the physical mixture group, the peak plasma drug concentration and area under the curve for drug delivery increased in the solid dispersion group, indicating that the process of this invention can improve the oral absorption level of the drug.

[0118] Monomeric compounds have rigid structures and high lattice energies, making dissolution difficult in the gastrointestinal environment. Hot-melt extrusion transforms drug molecules into an amorphous state, removing the lattice energy barrier to dissolution. The polymer carrier inhibits recrystallization, maintaining a supersaturated concentration. This altered apparent solubility increases the probability of the drug entering the bloodstream. Solid dispersion formulations enhance blood drug concentrations to meet the concentration thresholds required for drug penetration into cells, inhibition of p65 nuclear translocation, and binding of inflammatory factors in the interstitial space.

[0119] Test Example 6: Accelerated Stability Test of Solid Dispersions This test case aims to investigate the physical stability of solid dispersions prepared by hot melt extrusion under high temperature and high humidity conditions, and to assess whether amorphous drugs will recrystallize during storage, thereby affecting dissolution performance. The specific experimental steps are as follows: 1. Spread the prepared solid dispersion powder (taking the product of Example 2 as an example) evenly in an uncovered glass petri dish and place it in a constant temperature and humidity test chamber with a temperature of 40±2℃ and a relative humidity of 75±5%. The observation period is set to 6 months.

[0120] 2. Samples were taken from the constant temperature and humidity chamber at the end of month 0 (initial), month 1, month 3 and month 6 respectively for subsequent crystal form analysis and dissolution test.

[0121] 3. Take appropriate amounts of powder samples from different time points and scan them using an X-ray powder diffractometer (XRD). The tube voltage is 40 kV, the tube current is 40 mA, the scanning range 2θ is set to 5° to 40°, and the scanning step size is 0.02°. Record the X-ray diffraction patterns of the samples, and calculate the relative crystallinity of aloe-emodin, kaempferol, and quercetin in the system using integral analysis software.

[0122] 4. Conduct in vitro dissolution tests according to the paddle method in the dissolution determination method of the Chinese Pharmacopoeia. Accurately weigh solid dispersion samples at each time point, equivalent to approximately 50 mg of the total drug amount, and put them into a dissolution vessel containing 900 mL of degassed pH 6.8 phosphate buffer. The temperature of the dissolution medium should be kept constant at 37 ± 0.5℃, and the stirring speed should be set to 75 rpm.

[0123] 5. At the 45-minute mark of the dissolution process, use a syringe with a filter tip to draw 5 mL of the dissolution solution and add an equal volume of blank medium at the same temperature to the dissolution vessel. Filter the dissolution solution through a 0.45 μm microporous membrane and collect the filtrate. Determine the concentrations of the three drug components in the filtrate using high-performance liquid chromatography (HPLC) and calculate their cumulative dissolution rates at 45 minutes.

[0124] Table 6. Relative crystallinity and cumulative dissolution test data of solid dispersions under accelerated testing conditions

[0125] According to Table 6 and Figure 10 ( Figure 10 The graph shows the results of the physical stability test of the solid dispersion at 40℃ and 75% relative humidity (including two sub-graphs showing the changing trends of different indicators). At month 0, the relative crystallinity of aloe-emodin, kaempferol and quercetin were 0.81%, 1.12% and 0.95%, respectively, and the cumulative dissolution rate after 45 minutes was above 85%.

[0126] After six months of accelerated observation, the relative crystallinity of the three components increased (e.g., Figure 10 Sub-figure a shows a bar chart of relative crystallinity changes (with specific values ​​labeled), but the highest value at the end of the 6th month remained below 2.63%, showing no tendency to transform into a crystalline state; meanwhile, the cumulative dissolution rate decreased at 45 minutes (e.g., Figure 10 Sub-figure b shows a line graph of cumulative dissolution rate changes; the test results after 6 months remained above 80%. Figure 10 The overall trend shows that the relative crystallinity remained at a low level during the 6-month accelerated observation period; the cumulative in vitro dissolution decreased but remained at a high level overall, indicating that the amorphous drug system did not undergo recrystallization aging during storage.

[0127] Amorphous drugs exist in a high thermodynamic energy state and are prone to recrystallization when temperature and humidity rise. During extrusion, drug molecules are uniformly dispersed within the polymer network structure. After cooling and molding, the steric hindrance of the polymer matrix restricts thermal motion; the carrier molecules and monomer structure form a hydrogen bond network. Steric hindrance and chemical interactions solidify the amorphous conformation of the drug molecules, inhibiting the thermodynamic pathways for crystal nucleation. Data under accelerated conditions demonstrate that this formulation structure ensures the bioavailability of the drug composition within its shelf life, providing a basis for maintaining blood drug concentrations in vivo.

Claims

1. A hepatoprotective drug composition for relieving hangovers by regulating liver inflammation through the NF-κB pathway, characterized in that, It is produced by hot melt extrusion reaction of raw materials containing the following parts by weight in a co-rotating twin-screw extruder: Quercetin 6.67 to 13.34 parts, Kaempferol 6.67 to 13.33 parts, Aloe-emodin 6.66 to 13.33 parts, HPMCAS-HF polymer 40.0 to 60.0 parts, L-arginine 10.0 to 20.0 parts, Poloxamer 188 3.0 to 8.0 parts, PEG 400 2.0 to 5.0 parts, Deionized water 1.13 to 4.0 parts; The quercetin, kaempferol, and aloe-emodin together constitute the core active monomers of the composition; The HPMCAS-HF type polymer serves as a backbone network polymer and encapsulates the core active monomer during hot melt extrusion to form an amorphous dispersion system. The L-arginine acts as a microenvironment regulator and modulates the microscopic physicochemical environment of the core active monomer within the system. Poloxamer 188 acts as a compatibility wetting agent to promote the uniform dispersion of the active substance within the skeletal network. The PEG 400 and the deionized water form a transient mass transfer fluid, which is continuously injected into the hot melt extrusion mixing section to promote the full mixing and reaction of solid and liquid materials. Finally, the mixture is cooled and solidified to form an amorphous powder composition.

2. The hangover-relieving and liver-protecting drug composition according to claim 1, characterized in that, The mass fraction of the raw material is: The composition includes 10.0 parts of quercetin, 10.0 parts of kaempferol, 10.0 parts of aloe-emodin, 50.0 parts of HPMCAS-HF polymer, 15.0 parts of L-arginine, 5.0 parts of poloxamer 188, 3.5 parts of PEG 400, and 2.33 parts of deionized water.

3. The hangover-relieving and liver-protecting drug composition according to claim 1, characterized in that, The PEG 400 and the deionized water are mechanically mixed evenly at an ambient temperature of 20°C to 25°C and then allowed to stand at room temperature to degas and eliminate internal microbubbles, forming a uniform, transparent and colorless transient mass transfer fluid.

4. The hangover-relieving and liver-protecting drug composition according to claim 1, characterized in that, The quercetin, kaempferol, aloe-emodin, HPMCAS-HF polymer, and L-arginine were pre-screened using an 80-mesh industrial stainless steel sieve. For the waxy form of poloxamer 188, coarse sieving using a 20-mesh sieve is employed to de-agglomerate it. All sieved solid materials are mixed and homogenized in a clean room with a relative humidity of no more than 40% to form a physically dry powder.

5. The hangover-relieving and liver-protecting drug composition according to claim 1, characterized in that, The preparation process includes the following steps: According to the preset mass proportions of the formula, the solid materials in the raw materials are sieved and then fed into a three-dimensional motion mixer for mixing to obtain premixed powder. The PEG 400 and the deionized water were stirred to dissolve each other and allowed to stand to degas before being loaded into a liquid phase metering pump for later use. The premixed powder is fed into the co-rotating twin-screw extruder at a constant feeding rate and passes through the feeding zone, conveying zone, and initial melting zone in sequence. After the premixed powder enters the mixing zone, the liquid phase metering pump continuously injects the liquid phase additive into the barrel and the internal mixing thread element generates pressure inside the mixing zone to promote the full mixing and reaction of solid and liquid materials. After the mixture reaction, the material then enters the devolatilization zone and the vacuum pump is turned on to make the absolute pressure of the devolatilization zone negative to remove the moisture in the system and perform thermal compensation heating to maintain the fluidity of the melt. After devolatilization, the melt is extruded in strip form through the extrusion zone and die, and then quickly enters the traction cold roller system or pressure roller system for cooling and solidification at a constant cooling rate. Finally, the solidified material is pulverized and sieved to obtain the amorphous composition powder.

6. The hangover-relieving and liver-protecting drug composition according to claim 5, characterized in that, In the process of preparing the premixed powder, the solid material is fed into the mixing cylinder of the three-dimensional motion mixer and the total loading coefficient of the material is controlled to be between 40% and 50% of the total volume of the mixing cylinder. The operating speed of the three-dimensional motion mixer is set to 15 to 25 revolutions per minute and the mixing time is set to 20 to 30 minutes. After the mixing process is completed, the machine is stopped and left to stand still to allow the dust to settle.

7. The hangover-relieving and liver-protecting drug composition according to claim 5, characterized in that, During the operation of adding the premixed powder to the co-rotating twin-screw extruder and sequentially passing through the front section, the feeding rate of the premixed powder is controlled between 2.0 and 5.0 kg per hour; Set the screw speed to 200 to 250 revolutions per minute; The temperature of the feeding zone is set between 20°C and 30°C, the temperature of the conveying zone is set between 70°C and 90°C, and the temperature of the initial melting zone is set between 115°C and 125°C.

8. The hangover-relieving and liver-protecting drug composition according to claim 5, characterized in that, During the process of the premixed powder entering the mixing zone, the cylinder temperature of the mixing zone is set to be between 135°C and 145°C. The liquid phase additive is continuously injected into the barrel using the liquid phase metering pump, and the system pressure reaches between 1.5 MPa and 2.0 MPa.

9. The hangover-relieving and liver-protecting drug composition according to claim 5, characterized in that, During the process of the mixed reaction material entering the devolatilization zone, the absolute pressure of the devolatilization zone is controlled between -0.098 MPa and -0.090 MPa for exhaust and dehumidification. The heating compensation temperature of the devolatilization zone is set to be between 135°C and 140°C.

10. The hangover-relieving and liver-protecting drug composition according to claim 5, characterized in that, During the extrusion and cooling curing operation, the temperature of the extrusion zone and the die is set between 130°C and 135°C. After being extruded, the melt enters the traction cooling roller system or the pressure roller system, with the surface temperature set between 5°C and 10°C. The cooling rate is controlled at 50°C to 80°C per second for cooling and solidification, and the final solidified material is crushed and passed through a 60 to 80 mesh sieve.

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