High-stability spleen amino acid peptide oral freeze-dried powder and preparation method thereof

Abstract

The application discloses a high-stability spleen amino acid peptide oral lyophilized powder and a preparation method thereof, and relates to the technical fields of biochemical pharmacy and polypeptide preparation. The lyophilized powder comprises a spleen amino acid peptide stock solution, a trehalose-arginine conjugate, vitamin E phosphate ester wrapped by hydroxypropyl-beta-cyclodextrin, a cationic oligopeptide, a glycyrrhizic acid-chitooligosaccharide covalent conjugate and mannitol. Through the synergistic effect among the formula components, the lyophilization and storage process is effectively inhibited, the stability of the active compound after reconstitution is significantly improved, the oxidation degradation rate is greatly reduced, the dual functions of natural flavoring and oral mucosa adhesion are realized, the retention time of the active ingredient in the oral cavity is prolonged, and therefore the chemical stability, the physical stability and the bioactivity maintenance rate of the product are comprehensively improved. The used auxiliary materials are safe and reliable, and are suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the fields of biochemical pharmaceuticals and polypeptide preparations, specifically to a highly stable oral lyophilized spleen peptide and its preparation method. Background Technology

[0002] Spleen peptides are nucleotide and polypeptide complexes extracted from the spleen of healthy mammals. They possess biological activities such as immunomodulation and enhancing the body's resistance, and are clinically used as adjunctive treatment for recurrent respiratory infections and immunodeficiency. Oral lyophilized powder formulations are one of the main dosage forms of spleen peptides due to their portability, ease of administration, and avoidance of polypeptide degradation in liquid conditions.

[0003] However, existing oral lyophilized spleen peptide powders face the following technical challenges in preparation and storage. First, spleen peptide molecules contain two active components: polypeptides and nucleotides. During lyophilization, these components are prone to phase separation due to ice crystal formation and dehydration stress. This phase separation results in heterogeneous distribution of polypeptides and nucleotides in the lyophilized solid state, making it impossible for the active complex to reform after reconstitution, leading to a significant decrease in immunomodulatory activity. While conventional lyophilization protectants such as mannitol and sucrose can partially inhibit protein aggregation, they lack specific regulatory capabilities for the phase separation of polypeptide-nucleotide complexes. Second, spleen peptides are susceptible to oxidative degradation during long-term storage. Residual metal ions and unsaturated lipids in spleen peptide extracts can still catalyze oxidation reactions under low-temperature storage conditions, leading to the oxidation of methionine residues and nucleotide ring opening, generating inactive or sensitizing products. Existing technologies often employ nitrogen filling or vacuum packaging to delay oxidation, but oxidation occurs rapidly after packaging damage, lacking formulation-level antioxidant protection. Third, spleen peptides have an inherent fishy and bitter taste, resulting in poor oral compliance. To mask unpleasant odors, existing products often add fruit flavorings and high-intensity sweeteners. However, aldehydes and ketones in flavorings can undergo Maillard reactions with the amino groups of peptides, leading to decreased activity and the production of colored products; high-intensity sweeteners such as sucralose may induce phase separation during freeze-drying. Developing a natural flavoring system that does not rely on flavorings while avoiding interactions with active components is key to improving product quality. Fourth, existing oral lyophilized spleen peptide powders have a short residence time after dissolving in the oral cavity, and the active ingredient is quickly swallowed into the stomach, resulting in low bioavailability after degradation by gastric acid. Prolonging the residence time of the formulation on the oral mucosa to achieve local immune modulation and absorption via the oral mucosa is of positive significance for improving the efficacy of spleen peptides, but current technologies lack relevant designs.

[0004] Therefore, developing a highly stable oral lyophilized spleen peptide that can simultaneously address phase separation inhibition, antioxidant protection, flavoring without fragrance, and prolonged mucosal adhesion has significant clinical value and industrialization implications. Summary of the Invention

[0005] The purpose of this invention is to provide a highly stable oral lyophilized spleen peptide and its preparation method, so as to solve the problems mentioned in the background art.

[0006] In a first aspect, the present invention provides a highly stable oral lyophilized powder of spleen peptide, comprising the following raw materials in parts by weight: Spleen peptide stock solution (calculated as polypeptide) 50-100 parts; 30-60 parts of trehalose-arginine conjugate; 20-40 parts of vitamin E phosphate encapsulated in hydroxypropyl-β-cyclodextrin; 5-15 parts of cationic oligopeptides; 10-25 parts of glycyrrhizic acid-chitosan oligosaccharide covalent conjugate; Mannitol 100-200 parts; Add water for injection to a total of 1000 parts.

[0007] As a preferred embodiment of the present invention, the preparation method of the spleen peptide stock solution is as follows: Fresh pig spleen was removed of fat and connective tissue, homogenized, and then subjected to two freeze-thaw cycles. It was then hydrolyzed by protease and nuclease in sequence. After the enzymatic hydrolysis was completed, the enzyme was inactivated by heating at 95℃ for 10 min. The mixture was then purified by centrifugation, ultrafiltration, and preparative high performance liquid chromatography. The fraction with a molecular weight of 2000-4000 Da was collected and concentrated to a polypeptide content of 50-100 mg / mL.

[0008] As a preferred embodiment of the present invention, the preparation method of the trehalose-arginine conjugate is as follows: By weight, 100 parts of trehalose were dissolved in 600 parts of anhydrous dimethyl sulfoxide, 80 parts of 2,2,6,6-tetramethylpiperidin-1-oxy and 10 parts of sodium hypochlorite were added, and the reaction was carried out at room temperature for 2 hours to selectively oxidize the 6-position primary hydroxyl group of trehalose to a carboxyl group; then 85 parts of arginine were added and stirred until completely dissolved; the reaction system was cooled to 0-4℃, 120 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 120 parts of N-hydroxysuccinimide were added, and the reaction was carried out at room temperature for 24 hours; the reaction solution was poured into 3000 parts of anhydrous ethanol to precipitate, the precipitate was collected by centrifugation, washed 3 times with anhydrous ethanol, and dried under vacuum to obtain the final product.

[0009] As a preferred embodiment of the present invention, the method for preparing the hydroxypropyl-β-cyclodextrin-encapsulated vitamin E phosphate is as follows: By weight, 30 parts of hydroxypropyl-β-cyclodextrin were dissolved in 500 parts of purified water, and 10 parts of vitamin E phosphate were added. The mixture was ultrasonically dispersed at 60°C for 30 min. The temperature was then lowered to 25°C and stirred at 500 r / min for 24 h. The solution was freeze-dried to obtain a white, loose powder.

[0010] As a preferred embodiment of the present invention, the cationic oligopeptide is a polyarginine nonapeptide with the sequence structure Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg, which is prepared by solid-phase polypeptide synthesis and has a purity of not less than 95%.

[0011] As a preferred embodiment of the present invention, the preparation method of the glycyrrhizic acid-chitosan oligosaccharide covalent conjugate is as follows: By weight, 20 parts of chitosan oligosaccharide were dissolved in 500 parts of 0.1 mol / L 2-(N-morpholino)ethanesulfonic acid buffer and the pH was adjusted to 5.5; 10 parts of glycyrrhizic acid were dissolved in 100 parts of anhydrous ethanol, and 15 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 15 parts of N-hydroxysuccinimide were added and stirred at room temperature for 30 min; the activated solution was slowly added dropwise to the above chitosan oligosaccharide solution and stirred at room temperature for 12 h; the reaction solution was poured into 3000 parts of anhydrous ethanol to precipitate, the precipitate was collected by centrifugation and washed 3 times with anhydrous ethanol; the precipitate was dissolved in purified water and placed in a dialysis bag with a molecular weight cutoff of 500 Da, dialyzed with purified water for 48 h, and freeze-dried to obtain the final product; The weight-average molecular weight of the chitosan oligosaccharide is 1500 Da, and the dispersity is ≤1.5.

[0012] As a preferred embodiment of the present invention, the mannitol has a heavy metal content of no more than 5 ppm and an endotoxin content of less than 10 EU / g.

[0013] It should be noted that this invention uses a trehalose-arginine conjugate as a freeze-drying protectant. Trehalose forms hydrogen bonds with the amide bonds of the polypeptide through its hydroxyl groups, replacing water molecules to maintain the native conformation of the peptide chain; the guanidinium group in the conjugated arginine residues can interact electrostatically and with the phosphate groups of the nucleotides through hydrogen bonding, keeping the polypeptide and nucleotide spatially adjacent in the freeze-dried solid state and inhibiting phase separation. Compared with using trehalose or arginine alone, the two functional groups in the conjugate molecule are spatially close, which can produce a synergistic fixation effect.

[0014] It should be noted that hydroxypropyl-β-cyclodextrin-encapsulated vitamin E phosphate acts as an antioxidant. Vitamin E phosphate is a water-soluble vitamin E derivative with dual functions of scavenging free radicals and chelating metal ions, and can inhibit the oxidation of methionine and nucleotides in spleen peptides. Hydroxypropyl-β-cyclodextrin encapsulates the tocopherol moiety of vitamin E phosphate through a hydrophobic cavity, improving its dispersion stability in aqueous solution and forming an amorphous protective matrix after lyophilization, allowing for the slow release of vitamin E phosphate during storage and providing long-term antioxidant protection.

[0015] It should be noted that the cationic oligopeptide is a polyarginine nonapeptide. The lysine residues in this oligopeptide are positively charged and can electrostatically self-assemble with the negatively charged nucleotides in the spleen peptide to form a metastable complex. This complex pre-organizes the peptide and nucleotides non-covalently during lyophilization, reducing the stress-induced phase separation driving force. The arginine-glycine-aspartic acid sequence can mimic adhesion signals in the extracellular matrix, suggesting that it may promote the transient adhesion of orally lyophilized powder to the oral mucosa, providing initial anchoring for subsequent mucosal adhesion systems.

[0016] It should be noted that the glycyrrhizic acid-chitosan oligosaccharide covalent conjugate possesses both flavor-enhancing and mucosal adhesion functions. Chitosan oligosaccharide is a natural cationic polysaccharide that can penetrate the mucus layer on the oral mucosa and interact electrostatically with the mucosal epithelial cells, prolonging the retention time of the formulation in the oral cavity. Glycyrrhizic acid is a natural sweetener, approximately 150 times sweeter than sucrose, effectively masking the fishy and bitter taste of spleen peptides. It also lacks aldehyde and ketone structures and does not undergo Maillard reactions with peptides. Glycyrrhizic acid also possesses anti-inflammatory and immunomodulatory activities, exhibiting synergistic effects with spleen peptides. Covalently grafting glycyrrhizic acid onto chitosan oligosaccharide allows for synergistic effects, avoiding the migration and crystallization risks associated with small-molecule sweeteners while enhancing the mucosal adhesion ability of chitosan oligosaccharide.

[0017] It should be further noted that there is a synergistic effect between the trehalose-arginine conjugate, the cationic oligopeptide, and the glycyrrhizic acid-chitosan oligosaccharide covalent conjugate. The trehalose-arginine conjugate pre-organizes the peptides and nucleotides through guanidino-phosphate interaction, and the cationic oligopeptide further enhances the stability of this pre-organized structure through electrostatic cross-linking. Together, they construct a lyophilization protection network for the peptide-nucleotide complex. In the glycyrrhizic acid-chitosan oligosaccharide covalent conjugate, the chitosan oligosaccharide and the polyarginine segment of the cationic oligopeptide are both cationic polymers. They can competitively bind to the mucosal surface during oral dissolution, prolonging the local retention time of spleen peptide through synergistic adsorption. In addition, the hydrophobic aglycone portion of glycyrrhizic acid can interact hydrophobically with the hydrophobic region of vitamin E phosphate encapsulated by hydroxypropyl-β-cyclodextrin, forming antioxidant microdomains, further improving the dispersion stability of vitamin E phosphate in the lyophilized powder.

[0018] A second aspect of the present invention provides a method for preparing a highly stable oral lyophilized powder of spleen peptide, comprising the following steps: S1. Add the prescribed amount of spleen peptide stock solution to the mixing tank and heat to 35-40℃; S2. Add the formulated amounts of trehalose-arginine conjugate, hydroxypropyl-β-cyclodextrin-encapsulated vitamin E phosphate, cationic oligopeptide, glycyrrhizic acid-chitosan oligosaccharide covalent conjugate and mannitol to the solution obtained in S1 in sequence. Stir until completely dissolved, add water for injection to the total volume, and continue stirring for 15 minutes to obtain spleen peptide solution. S3. The spleen peptide solution obtained in S2 is filtered through a 0.22μm filter membrane for sterilization, and dispensed into vials, 1-2mL per vial, and half-stopped. S4. Place the half-filled vials obtained in S3 into a freeze dryer and freeze dry them according to the following freeze-drying curve: In the pre-freezing stage, lower the temperature of the separator to -45°C within 1 hour and keep it at that temperature for 2 hours; In the first drying stage, raise the temperature of the separator to -25°C within 2 hours, set the condenser temperature, and keep it at that temperature for 16 hours under a vacuum of less than 20 Pa; In the second drying stage, raise the temperature of the separator to 25°C within 2 hours and keep it at that temperature for 6 hours under a vacuum of less than 10 Pa. S5. After the secondary drying is completed, high-purity nitrogen gas is introduced into the freeze-drying chamber to atmospheric pressure, and then the chamber is plugged and capped to obtain the product.

[0019] As a preferred embodiment of the present invention, the condenser temperature in the primary drying stage of step S4 is set to below -60°C.

[0020] As a preferred embodiment of the present invention, the ultimate vacuum degree of the secondary drying stage in step S4 does not exceed 5 Pa.

[0021] It should be noted that the temperature setting in step S4 for the primary drying stage is based on the following: the collapse temperature of the product is measured to be -22℃±1℃ using a freeze-drying microscope, and the temperature of the primary drying shelf is set to -25℃ to ensure that the product temperature remains below the collapse temperature throughout the drying process.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves the pre-organization and fixation of polypeptides and nucleotides in spleen peptide in freeze-dried solid state through the synergistic effect of trehalose-arginine conjugate and cationic oligopeptide, effectively inhibiting phase separation during freeze-drying and storage, and significantly improving the immunogenicity retention rate of the active complex after reconstitution.

[0023] (2) The present invention provides long-lasting antioxidant protection through vitamin E phosphate encapsulated by hydroxypropyl-β-cyclodextrin, and forms antioxidant microdomains with the hydrophobic region of the glycyrrhizic acid-chitosan oligosaccharide covalently coupled, which further enhances the oxidative stability of the formulation system and significantly reduces the degradation rate of spleen peptide in accelerated tests.

[0024] (3) This invention achieves the dual functions of natural flavoring and oral mucosal adhesion through glycyrrhizic acid-chitosan oligosaccharide covalent coupling, without the need to add fruit flavorings and high-concentration synthetic sweeteners, avoiding Maillard reaction and phase separation risks, while prolonging the retention time of active ingredients in the oral cavity, which helps to improve the absorption efficiency through the oral mucosa and the local immune regulation effect.

[0025] (4) All components of this invention are food contact safety grade or pharmaceutical excipient grade, and no toxic organic solvents, heavy metal catalysts or artificial flavorings are added. It meets the safety requirements of oral preparations and has good prospects for industrial application. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The sources of some components in the examples and comparative examples are as follows: Table 1

[0028] Preparation Example 1 The preparation steps of the trehalose-arginine conjugate are as follows: By weight, 100 parts trehalose and 85 parts arginine were dissolved in 600 parts anhydrous dimethyl sulfoxide and stirred until completely dissolved. 120 parts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 120 parts N-hydroxysuccinimide were added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was poured into 3000 parts anhydrous ethanol to precipitate the precipitate. The precipitate was collected by centrifugation, washed three times with anhydrous ethanol, and dried under vacuum at 50°C for 12 hours to obtain the final product.

[0029] Preparation Example 2 The preparation steps of vitamin E phosphate encapsulated by hydroxypropyl-β-cyclodextrin are as follows: Dissolve 30 parts by weight of hydroxypropyl-β-cyclodextrin in 500 parts of purified water, add 10 parts of vitamin E phosphate, and sonicate at 60°C for 30 min. Cool to 25°C and stir at 500 r / min for 24 h. Freeze-dry the solution to obtain a white, loose powder, which is the product.

[0030] Preparation Example 3 The preparation steps of glycyrrhizic acid-chitosan oligosaccharide covalent conjugate are as follows: By weight, 20 parts of chitosan oligosaccharide were dissolved in 500 parts of 0.1 mol / L 2-(N-morpholino)ethanesulfonic acid buffer, and the pH was adjusted to 5.5. 10 parts of glycyrrhizic acid were dissolved in 100 parts of anhydrous ethanol, and 15 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 15 parts of N-hydroxysuccinimide were added. The mixture was stirred and activated at room temperature for 30 min. The activated solution was slowly added dropwise to the chitosan oligosaccharide solution, and the reaction was stirred at room temperature for 12 h. The reaction solution was poured into 3000 parts of anhydrous ethanol to precipitate the precipitate. The precipitate was collected by centrifugation and washed three times with anhydrous ethanol. The precipitate was dissolved in purified water and placed in a dialysis bag with a molecular weight cutoff of 500 Da. Dialysis was performed with purified water for 48 h, changing the water every 6 h. After dialysis, the precipitate was freeze-dried to obtain the final product. Example 1

[0031] A highly stable spleen peptide oral lyophilized powder, comprising the following ingredients by weight: 75 portions of spleen peptide stock solution (calculated as polypeptide); 45 parts of trehalose-arginine conjugate (prepared in Example 1); 30 parts of hydroxypropyl-β-cyclodextrin-encapsulated vitamin E phosphate (prepared in Example 2); 10 portions of cationic oligopeptide (Arg-Gly-Asp)3-Lys8; 18 parts of glycyrrhizic acid-chitosan oligosaccharide covalent conjugate (prepared in Example 3); 150 parts mannitol; Add water for injection to a total of 1000 parts; The preparation method of the spleen peptide stock solution is as follows: Fresh pig spleen was removed of fat and connective tissue, homogenized, and then subjected to two freeze-thaw cycles. It was then hydrolyzed by protease and nuclease in sequence. After the enzymatic hydrolysis was completed, the enzyme was inactivated by heating at 95℃ for 10 min. The mixture was then purified by centrifugation, ultrafiltration, and preparative high performance liquid chromatography. The fraction with a molecular weight of 2000-4000 Da was collected and concentrated to a polypeptide content of 80 mg / mL.

[0032] The preparation method is as follows: S1. Add the spleen peptide stock solution to the mixing tank and heat it to 38°C; S2. Add trehalose-arginine conjugate, vitamin E phosphate encapsulated by hydroxypropyl-β-cyclodextrin, cationic oligopeptide, glycyrrhizic acid-chitosan oligosaccharide covalent conjugate and mannitol to the solution obtained in S1 in sequence, stir until completely dissolved, add water for injection to the total volume, and continue stirring for 15 min to obtain spleen peptide solution. S3. The spleen peptide solution obtained in S2 was sterilized by filtration through a 0.22μm polyethersulfone filter membrane, and dispensed into 10mL vials, each vial containing 1.5mL and partially stoppered. S4. Place the half-filled vials obtained in S3 into a freeze dryer and freeze dry them according to the following freeze-drying curve: In the pre-freezing stage, lower the temperature of the separator to -45℃ within 1 hour and keep it at that temperature for 2 hours; In the first drying stage, raise the temperature of the separator to -20℃ within 2 hours, set the condenser temperature to -60℃, the vacuum degree to less than 20Pa, and keep it at that temperature for 12 hours; In the second drying stage, raise the temperature of the separator to 25℃ within 2 hours, the ultimate vacuum degree to no more than 5Pa, and keep it at that temperature for 6 hours. S5. After the secondary drying is completed, high-purity nitrogen gas is introduced into the freeze-drying chamber to atmospheric pressure, and then the chamber is plugged and capped to obtain the product. Example 2

[0033] The difference between this embodiment and Example 1 is that the weight parts of the raw materials are adjusted as follows: 50 parts of spleen peptide stock solution (calculated as polypeptide), 30 parts of trehalose-arginine conjugate, 20 parts of vitamin E phosphate encapsulated with hydroxypropyl-β-cyclodextrin, 5 parts of cationic oligopeptide, 10 parts of glycyrrhizic acid-chitosan oligosaccharide covalent conjugate, 100 parts of mannitol, and water for injection to 1000 parts. The remaining preparation methods and lyophilization conditions are the same as in Example 1. Example 3

[0034] The difference between this embodiment and Example 1 is that the weight parts of the raw materials are adjusted as follows: 100 parts of spleen peptide stock solution (calculated as polypeptide), 60 parts of trehalose-arginine conjugate, 40 parts of vitamin E phosphate encapsulated with hydroxypropyl-β-cyclodextrin, 15 parts of cationic oligopeptide, 25 parts of glycyrrhizic acid-chitosan oligosaccharide covalent conjugate, 200 parts of mannitol, and water for injection is increased to 1000 parts. The remaining preparation methods and lyophilization conditions are the same as in Example 1.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that the trehalose-arginine conjugate prepared in Example 1 was not added; instead, an equal amount of trehalose was added. All other raw materials and preparation methods are the same as in Example 1.

[0036] Comparative Example 2 The difference between this comparative example and Example 1 is that the hydroxypropyl-β-cyclodextrin-encapsulated vitamin E phosphate prepared in Example 2 was not added; instead, an equal part by weight of unencapsulated vitamin E phosphate was added. The remaining raw materials and preparation methods are the same as in Example 1.

[0037] Comparative Example 3 The difference between this comparative example and Example 1 is that the glycyrrhizic acid-chitosan oligosaccharide covalent conjugate prepared in Example 3 was not added; instead, an equal weight mixture of chitosan oligosaccharide and glycyrrhizic acid (chitosan oligosaccharide to glycyrrhizic acid weight ratio 2:1) was added. The remaining raw materials and preparation methods are the same as in Example 1.

[0038] Comparative Example 4 The difference between this comparative example and Example 1 is that no cationic oligopeptide was added, while the other raw materials and preparation methods are the same as in Example 1.

[0039] test: I. Phase Separation Degree Test The glass transition temperature and cold crystallization behavior of the lyophilized powders were determined using differential scanning calorimetry (DSC). 5 mg of each of the lyophilized powders prepared in Examples 1-3 and Comparative Examples 1-4 were placed in sealed aluminum crucibles and scanned from -50°C to 50°C at a heating rate of 10°C / min, and the heat flow curves were recorded. The degree of phase separation was expressed as the percentage of the peak area of ​​the cold crystallization peak to the total heat flow change; a higher percentage indicated more severe phase separation.

[0040] II. Test of the formation rate of active complex after reconstitution One vial each of the lyophilized powders prepared in Examples 1-3 and Comparative Examples 1-4 were reconstituted with 2 mL of purified water. The aggregation state of the reconstituted spleen peptide was determined by size exclusion high-performance liquid chromatography (HPLC). The chromatographic column was a TSK-GEL G2000SWXL, the mobile phase was 0.1 mol / L phosphate buffer at pH 7, the flow rate was 0.5 mL / min, and the detection wavelengths were 214 nm and 260 nm. The formation rate of the active complex was expressed as the ratio of the peak area with consistent retention times at both wavelengths (214 nm and 260 nm) to the total peak area. The active complex was defined as a chromatographic component with a molecular weight range of 3000-4000 Da and an UV absorbance ratio of 1.2-1.8 at 214 nm and 260 nm.

[0041] III. Oxidative Stability Test The content of methionine sulfoxide in lyophilized powder was determined by high performance liquid chromatography (HPLC). 10 mg each of the lyophilized powders prepared in Examples 1-3 and Comparative Examples 1-4 were dissolved in 1 mL of purified water, and an equal volume of 0.1 mol / L methanesulfonic acid was added for acid hydrolysis. After hydrolysis, the samples were derivatized with o-phthalaldehyde before injection. A C18 column (250 mm × 4.6 mm, 5 μm) was used, and the mobile phase was acetonitrile:water (65:35, containing 0.1% trifluoroacetic acid). The excitation wavelength for fluorescence detection was 340 nm, and the emission wavelength was 455 nm. The degree of oxidation was expressed as the ratio of the peak area of ​​methionine sulfoxide to the total peak area of ​​methionine and methionine sulfoxide.

[0042] IV. Taste and Flavoring Effect Test Twenty healthy volunteers (aged 22-35, half male and half female, selected for a phenylthiourea taste test to ensure normal taste sensitivity) were selected. A double-blind method was used to evaluate the taste of the freeze-dried powders prepared in Examples 1-3 and Comparative Examples 1-4. All samples were coded by a third party and placed in uniform opaque containers. Evaluators were completely isolated from the sample preparers. The freeze-dried powder was dissolved directly on the tongue. After each evaluation, the mouth was rinsed with purified water, and the next evaluation was conducted after a 30-minute interval. Evaluation indicators included bitterness intensity (0-10 points, higher scores indicate stronger bitterness), fishy odor intensity (0-10 points, higher scores indicate stronger fishy odor), and overall acceptability (0-10 points, higher scores indicate better acceptability).

[0043] V. Oral Mucosal Adhesion Time Test The adhesion time was determined using an isolated porcine oral mucosa model. Fresh porcine buccal mucosa was taken and fixed onto the platform of a mucosal adhesion testing instrument. The lyophilized powders prepared in Examples 1-3 and Comparative Examples 1-4 were pressed into discs with a diameter of 5 mm and a thickness of 2 mm, weighed, and placed on the mucosal surface, with a pressure of 0.5 N applied for 30 s. The mucosa was fixed at a 30° tilt angle and rinsed with phosphate buffer at pH 6.8 at a flow rate of 1 mL / min. The time it took for the discs to completely detach from the mucosal surface was recorded.

[0044] VI. Accelerated Stability Test The lyophilized powders prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to accelerated testing under conditions of 40℃±2℃ and 75%±5% relative humidity, in accordance with the Guidelines for Stability Testing of Raw Materials and Preparations in General Chapter 9001 of the 2020 Edition of the Chinese Pharmacopoeia, Part IV. Samples were taken at 0, 1, 2, 3, and 6 months to determine the appearance, the rate of formation of active complex after reconstitution, the degree of oxidation, and the immunomodulatory activity of each sample.

[0045] VII. Summary of Results Table 2: Test results of phase separation degree and formation rate of active complex after resolution

[0046] Table 3: Results of Oxidation Degree Test

[0047] Table 4: Taste Evaluation Results

[0048] Table 5: Results of Oral Mucosal Adhesion Time Test

[0049] Table 6: Accelerated stability test results (6 months)

[0050] VIII. Discussion of Results As shown in Table 2, the phase separation degree of the spleen peptide oral lyophilized powder prepared in Examples 1-3 of this invention was significantly lower than that in Comparative Examples 1-4, and the formation rate of active complexes after reconstitution was close to or exceeded 90%. In Comparative Example 1, trehalose was used instead of the trehalose-arginine conjugate, resulting in a significantly increased phase separation degree and a substantial decrease in the formation rate of active complexes. This demonstrates that the trehalose-arginine conjugate achieves directional fixation of polypeptide-nucleotide complexes through the interaction between the guanidine group and phosphate and the hydrogen bonding with trehalose, with a significantly better effect than using trehalose alone. Comparative Example 4, without the addition of cationic oligopeptides, also showed a significantly higher phase separation degree than Example 1, and a decreased formation rate of active complexes, demonstrating a synergistic effect between the electrostatic pre-organization of cationic oligopeptides and the trehalose-arginine conjugate. Although the formation rates of active complexes in Comparative Examples 2 and 3 decreased, the magnitude was smaller than that in Comparative Example 1, indicating that phase separation was mainly synergistically regulated by the trehalose-arginine conjugate and the cationic oligopeptides.

[0051] Table 3 shows that the oxidation levels of Examples 1-3 remained low even after 6 months of accelerated testing. Comparative Example 2, using unencapsulated vitamin E phosphate instead of the hydroxypropyl-β-cyclodextrin inclusion complex, showed a significant increase in oxidation levels both initially and after accelerated testing. This demonstrates that hydroxypropyl-β-cyclodextrin inclusion significantly improves the dispersion stability and antioxidant efficiency of vitamin E phosphate in the lyophilized powder. The increased oxidation levels in Comparative Examples 1 and 3 compared to Example 1 are presumably related to the exposure of the active complex to an oxidizing environment due to phase separation, further illustrating that phase separation inhibition also indirectly contributes to oxidation protection.

[0052] Table 4 shows that the bitterness and fishy odor intensity of Examples 1-3 were significantly lower than those of Comparative Examples 1, 3, and 4. Comparative Example 1 exhibited the highest bitterness and fishy odor intensity among all comparative examples, presumably related to phase separation leading to product structural degradation and exposure of the active complex. Impaired structural integrity may have made the fishy odor components more easily released. Comparative Example 3 used a physical mixture of chitosan oligosaccharide and glycyrrhizic acid instead of the covalently coupled compound, resulting in a significant decrease in the flavor-correcting effect, an increase in bitterness and fishy odor intensity, and a reduction in overall acceptability. This demonstrates that the covalent coupling of glycyrrhizic acid and chitosan oligosaccharide plays a crucial role in the flavor-correcting function. It is speculated that the covalent coupling ensures uniform distribution of glycyrrhizic acid on the chitosan oligosaccharide backbone, avoiding localized crystallization or aggregation of glycyrrhizic acid during physical mixing, thus achieving a continuous and stable release of sweetness.

[0053] As shown in Table 5, the mucosal adhesion time of Examples 1-3 was significantly longer than that of Comparative Examples 3 and 4. Comparative Example 1 had the shortest adhesion time, worse than Comparative Example 4, which is presumably related to phase separation and product structure collapse caused by the absence of the trehalose-arginine conjugate. Impaired structural integrity may have weakened the physical adhesion effect. Comparative Example 3 used a physical mixture instead of the covalent coupling agent, resulting in a significant reduction in adhesion time, demonstrating that covalent coupling enhanced the hydrophobic anchoring effect of glycyrrhizic acid and improved the mucosal penetration ability of chitosan oligosaccharide. Comparative Example 4 did not add cationic oligopeptides, and its adhesion time was shorter than that of Example 1, but the reduction was less than that of Comparative Example 3. It is presumed that the cationic oligopeptides and the glycyrrhizic acid-chitosan oligosaccharide covalent coupling synergistically promote mucosal adhesion through different mechanisms; that is, the cationic oligopeptides provide initial electrostatic adsorption, while the glycyrrhizic acid-chitosan oligosaccharide covalent coupling provides long-term penetration and retention.

[0054] As shown in Table 6, after 6 months of accelerated testing, the appearance, active complex formation rate / oxidation degree, and immunomodulatory activity retention rate of Examples 1-3 were significantly better than those of Comparative Examples 1-4. Comparative Example 1 showed a light yellow color and slight collapse, suggesting that phase separation-induced product structural degradation may have accelerated the Maillard reaction or oxidative discoloration. Comparative Example 2 showed a sharp increase in oxidation degree and a significant decrease in the active complex formation rate, demonstrating that in the absence of effective antioxidant protection in the formulation, oxidative degradation and phase separation can mutually promote each other, accelerating product failure. Although the active complex formation rate of Comparative Example 3 was higher than that of Comparative Example 1, it was still significantly lower than that of Example 1, proving that physical mixtures cannot completely replace the function of covalently coupled compounds. After 6 months of accelerated testing, the immunomodulatory activity retention rate of Examples 1-3 was not less than 85%, while that of Comparative Examples 1-4 was less than 75%, demonstrating that the formulation system of the present invention effectively protects the immunomodulatory activity of spleen peptides and achieves the technical goal of high stability.

[0055] In summary, the highly stable spleen peptide oral lyophilized powder prepared in Examples 1-3 of this invention successfully overcomes the three major technical challenges of phase separation, oxidative degradation, and poor oral compliance in the prior art through the synergistic phase separation inhibition effect of trehalose-arginine conjugate and cationic oligopeptide, the long-lasting antioxidant effect of vitamin E phosphate encapsulated by hydroxypropyl-β-cyclodextrin, and the dual functions of natural flavoring and mucosal adhesion of glycyrrhizic acid-chitosan oligosaccharide covalent coupling. It exhibits excellent activity retention and physicochemical stability in accelerated stability tests and has good prospects for clinical application and industrialization.

[0056] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0057] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the scope defined by the invention, and all such modifications and additions should fall within the protection scope of the present invention.

Claims

1. A highly stable oral lyophilized powder of spleen peptide, characterized in that: Including the following parts by weight of raw materials: 50-100 parts of spleen peptide stock solution; 30-60 parts of trehalose-arginine conjugate; 20-40 parts of vitamin E phosphate encapsulated in hydroxypropyl-β-cyclodextrin; 5-15 parts of cationic oligopeptides; 10-25 parts of glycyrrhizic acid-chitosan oligosaccharide covalent conjugate; Mannitol 100-200 parts; Add water for injection to a total of 1000 parts.

2. The highly stable spleen peptide oral lyophilized powder according to claim 1, characterized in that: The preparation method of the spleen peptide stock solution is as follows: Fresh pig spleens were taken, and fat and connective tissue were removed. The mixture was homogenized and then repeatedly frozen and thawed twice. It was then purified by protease hydrolysis, nuclease hydrolysis, centrifugation, ultrafiltration, and preparative high-performance liquid chromatography. The fractions with molecular weights of 2000-4000 Da were collected and concentrated to a polypeptide content of 50-100 mg / mL.

3. The highly stable spleen peptide oral lyophilized powder according to claim 1, characterized in that: The preparation method of the trehalose-arginine conjugate is as follows: By weight, 100 parts of trehalose were dissolved in 600 parts of anhydrous dimethyl sulfoxide, 80 parts of 2,2,6,6-tetramethylpiperidin-1-oxy and 10 parts of sodium hypochlorite were added, and the reaction was carried out at room temperature for 2 hours to selectively oxidize the 6-position primary hydroxyl group of trehalose to a carboxyl group; then 85 parts of arginine were added and stirred until completely dissolved; the reaction system was cooled to 0-4℃, 120 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 120 parts of N-hydroxysuccinimide were added, and the reaction was carried out at room temperature for 24 hours; the reaction solution was poured into 3000 parts of anhydrous ethanol to precipitate, the precipitate was collected by centrifugation, washed 3 times with anhydrous ethanol, and dried under vacuum to obtain the final product.

4. The highly stable spleen peptide oral lyophilized powder according to claim 1, characterized in that: The method for preparing the hydroxypropyl-β-cyclodextrin-encapsulated vitamin E phosphate is as follows: By weight, 30 parts of hydroxypropyl-β-cyclodextrin were dissolved in 500 parts of purified water, and 10 parts of vitamin E phosphate were added. The mixture was ultrasonically dispersed at 60°C for 30 min. The temperature was then lowered to 25°C and stirred at 500 r / min for 24 h. The solution was freeze-dried to obtain a white, loose powder.

5. The highly stable spleen peptide oral lyophilized powder according to claim 1, characterized in that: The cationic oligopeptide is a polyarginine nonapeptide with the sequence structure Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg, prepared by solid-phase polypeptide synthesis, with a purity of not less than 95%.

6. The highly stable spleen peptide oral lyophilized powder according to claim 1, characterized in that: The preparation method of the glycyrrhizic acid-chitosan oligosaccharide covalent conjugate is as follows: By weight, 20 parts of chitosan oligosaccharide were dissolved in 500 parts of 0.1 mol / L 2-(N-morpholino)ethanesulfonic acid buffer and the pH was adjusted to 5.5; 10 parts of glycyrrhizic acid were dissolved in 100 parts of anhydrous ethanol, and 15 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 15 parts of N-hydroxysuccinimide were added and activated by stirring at room temperature for 30 min; the activated solution was added dropwise to the above chitosan oligosaccharide solution and the reaction was stirred at room temperature for 12 h; the reaction solution was poured into 3000 parts of anhydrous ethanol to precipitate, the precipitate was collected by centrifugation and washed 3 times with anhydrous ethanol; the precipitate was dissolved in purified water and placed in a dialysis bag with a molecular weight cutoff of 500 Da, dialyzed with purified water for 48 h, and freeze-dried to obtain the final product.

7. The highly stable spleen peptide oral lyophilized powder according to claim 6, characterized in that: The weight-average molecular weight of the chitosan oligosaccharide is 1500 Da, and the dispersity is ≤1.

5.

8. A method for preparing a highly stable oral lyophilized powder of spleen peptide as described in any one of claims 1-7, characterized in that: Includes the following steps: S1. Add the prescribed amount of spleen peptide stock solution to the mixing tank and heat to 35-40℃; S2. Add the formulated amounts of trehalose-arginine conjugate, hydroxypropyl-β-cyclodextrin-encapsulated vitamin E phosphate, cationic oligopeptide, glycyrrhizic acid-chitosan oligosaccharide covalent conjugate and mannitol to the solution obtained in S1 in sequence. Stir until completely dissolved, add water for injection to the total volume, and continue stirring for 15 minutes to obtain spleen peptide solution. S3. The spleen peptide solution obtained in S2 is filtered through a 0.22μm filter membrane for sterilization, and dispensed into vials, 1-2mL per vial, and half-stopped. S4. Place the half-filled vials obtained in S3 into a freeze dryer and freeze dry them according to the following freeze-drying curve: In the pre-freezing stage, lower the temperature of the separator to -45°C within 1 hour and keep it at that temperature for 2 hours; In the first drying stage, raise the temperature of the separator to -25°C within 2 hours, set the condenser temperature, and keep it at that temperature for 16 hours under a vacuum of less than 20 Pa; In the second drying stage, raise the temperature of the separator to 25°C within 2 hours and keep it at that temperature for 6 hours under a vacuum of less than 10 Pa. S5. After the secondary drying is completed, high-purity nitrogen gas is introduced into the freeze-drying chamber to atmospheric pressure, and then the chamber is plugged and capped to obtain the product.

9. The method for preparing a highly stable oral lyophilized spleen peptide powder according to claim 8, characterized in that: In step S4, the condenser temperature during the primary drying stage is set to below -60°C.

10. The method for preparing a highly stable oral lyophilized spleen peptide powder according to claim 8, characterized in that: In step S4, the ultimate vacuum degree of the secondary drying stage does not exceed 5 Pa.

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