Wood frog egg peptide microsphere preparation with enteric protection and preparation method of wood frog egg peptide microsphere preparation

Abstract

The invention discloses a wood frog egg peptide microsphere preparation with enteric protection and a preparation method of the wood frog egg peptide microsphere preparation, and belongs to the technical field of pharmaceutical preparations. The preparation comprises wood frog egg peptide subjected to antigen removal treatment, a biodegradable polymer (polylactic acid-glycolic acid copolymer, polylactic acid or chitosan), an enteric-coated material (acrylic resin, hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate) and an additive, and the mass ratio of the wood frog egg peptide to the biodegradable polymer is 1: (2-10). The dosage of the enteric-coated material is 5-20% of the total mass of the microspheres. The preparation method comprises the following steps: carrying out enzymolysis, ultrafiltration, gel chromatography purification and antigen removal treatment (affinity chromatography / chemical modification / ultrafiltration combined with gel chromatography) on wood frog egg peptide; preparing microspheres through an improved single emulsion-solvent evaporation method; and coating an enteric-coated material. The preparation disclosed by the invention is stable in drug release, high in bioavailability, low in immunogenicity, simple and controllable in preparation process and sustainable in raw material, and is suitable for enteric preparation application of wood frog egg peptide bioactive substances.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to a forest frog egg peptide microsphere formulation with enteric coating protection and its preparation method. Background Technology

[0002] With increasing public awareness of health, bioactive peptides have been widely researched and developed due to their unique physiological functions and low toxicity. Forest frog egg peptides, as a substance with various biological activities such as antioxidation, immune regulation, and anti-fatigue, have shown great application potential in the pharmaceutical and health product fields.

[0003] Currently, preparing frog egg peptides into microspheres and endowing them with enteric coating protection is one of the important means to improve their efficacy and stability. However, existing frog egg peptide microsphere preparations with enteric coating protection and their preparation methods have many problems.

[0004] In terms of formulation, firstly, production costs are high. The production of microsphere formulations requires highly specialized equipment and technology. Specialized emulsification and solidification equipment is indispensable in the preparation process, and precise control of various process parameters is essential, undoubtedly increasing production costs. Furthermore, for frog egg peptide microsphere formulations, the initial extraction and purification of frog eggs further increases costs. Secondly, drug release is unstable. The drug release rate of microsphere formulations is affected by various factors such as the size, shape, surface properties of the microspheres, and the release medium. In the intestinal environment, frog egg peptide microsphere formulations are interfered with by factors such as intestinal peristalsis and digestive fluid composition, leading to unstable egg peptide release rates and thus affecting efficacy. Thirdly, absorption is incomplete. The particle size and surface charge of the microspheres limit the absorption of frog egg peptide microsphere formulations in vivo, resulting in insufficient absorption of some drugs and reduced bioavailability. Additionally, there are immunogenicity issues. As a foreign substance, frog egg peptides may trigger immune responses after entering the human body, leading to adverse reactions such as allergies, affecting the safety and applicability of the formulation.

[0005] In terms of preparation methods, the process is cumbersome. Using methods similar to traditional peptide microspheres, such as double emulsification, involves multiple emulsification processes, solvent evaporation, or extraction. This not only prolongs the production cycle and increases costs but also introduces uncertainties such as uneven particle size distribution and unstable encapsulation efficiency in the final microspheres. Poor raw material sustainability is also a major issue. Forest frog eggs are typically obtained from forest frogs, and large-scale acquisition can impact frog survival and the ecological environment, hindering sustainable resource utilization. Furthermore, the quality and yield of forest frog eggs are limited by seasonal and geographical factors, affecting the stability of formulation production. Finally, quality control is challenging. The preparation process requires strict control over multiple indicators, including the quality of forest frog egg peptides, microsphere particle size, encapsulation efficiency, and drug loading. However, variations in raw material quality and fluctuations in process parameters lead to unstable product quality, increasing the difficulty of quality control.

[0006] However, existing technologies have the following shortcomings: CN105341907B merely masks the fishy smell of frog egg polypeptides with composite wall materials and does not involve "immunogenicity risk control" of pharmaceutical preparations; The patent for polypeptide microspheres, such as CN116869943A, does not address the specific immunogenicity of frog egg peptides. Furthermore, large molecules such as ovalbumin naturally present in frog eggs can easily trigger human immune responses, thus limiting their clinical application.

[0007] Therefore, developing a low-cost, stable, fully absorbed, low-immunogenic, simple, sustainable, and easily quality-controlled enteric-coated frog egg peptide microsphere formulation and its preparation method is of great practical significance. Summary of the Invention

[0008] The purpose of this invention is to provide a *Rana spp.* egg peptide microsphere formulation with enteric coating protection and its preparation method, thereby addressing the aforementioned problems in the prior art. Through innovative preparation processes and formulation design, production costs are reduced, drug release stability and absorption efficiency are improved, and immunogenicity is reduced. This is achieved by deantigenating the *Rana spp.* egg peptide (e.g., removing immunogenic proteins through affinity chromatography, chemically modifying to block antigenic epitopes), or removing large molecular immunogenic substances during extraction via ultrafiltration / gel chromatography, thus reducing the risk of immune responses triggered by the formulation. Simultaneously, the preparation process is simplified, improving the sustainability and quality control of raw materials. Artificially controlled breeding technology is employed, allowing for the rational collection of eggs during the *Rana spp.* breeding season (≥50% of eggs per female frog are retained for natural reproduction), avoiding overfishing of wild populations. Furthermore, the extraction process is optimized, increasing the utilization rate of *Rana spp.* eggs to over 90%. In the prior art, *Rana spp.* egg peptides, as polypeptides, face key challenges in formulation, including low bioavailability due to gastric acid degradation and immune responses triggered by large molecular proteins in natural components. This invention effectively solves these problems through enteric coating protection and deantigenation treatment.

[0009] The technical solution adopted by this invention to solve its technical problem is: a *Rana cristatum* ovum peptide microsphere formulation with enteric coating protection, comprising *Rana cristatum* ovum peptide, a biodegradable polymer, an enteric coating material, and optional additives; the *Rana cristatum* ovum peptide is subjected to antigen treatment; the biodegradable polymer is one or more of polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), and chitosan; the enteric coating material is one or more of acrylic resin, hydroxypropyl methylcellulose phthalate (HPMCP), and cellulose acetate phthalate (CAP); the mass ratio of *Rana cristatum* ovum peptide to biodegradable polymer is 1:(2-10), and the amount of enteric coating material is 5%-20% of the total mass of the microspheres. The frog egg peptides undergo antigen removal treatment, specifically including: Immunogenic proteins such as ovalbumin are specifically removed by affinity chromatography (such as Protein A / G chromatography columns), while bioactive peptide components are retained by utilizing the principle of antigen-antibody specific binding. Alternatively, ultrafiltration (with a molecular weight cutoff of 3000 Da) combined with gel chromatography (such as Sephadex G-25) can be used to remove large molecular weight proteins with a molecular weight >3000 Da while retaining small molecular weight peptides with low immunogenicity. Alternatively, the peptide can be chemically modified, for example through acylation (such as succinic anhydride modification of lysine residues), alkylation, or polyethylene glycolation, to block the antigenic epitopes on the surface of the peptide and prevent the immune system from recognizing them.

[0010] After the above treatment, the immunogenicity of frog egg peptide was significantly reduced. ELISA test showed that the antibody detection level of the treated preparation was reduced by more than 70% compared with the untreated group, effectively reducing the risk of immune response in vivo.

[0011] Specifically, the antioxidant is one or more of vitamin C, vitamin E, and propyl gallate.

[0012] Specifically, the plasticizer is one or more of triethyl citrate, diethyl phthalate, and polyethylene glycol.

[0013] Specifically, the surfactant is one or more of polyvinyl alcohol (PVA), Tween, and Span.

[0014] A method for preparing enteric-coated frog egg peptide microspheres includes the following steps: S1. Extraction and deantigenation of frog egg peptides: Wash fresh frog eggs, add phosphate buffer solution to homogenize, enzymatically hydrolyze, centrifuge, filter to remove impurities, and select the following processing methods according to the antigen removal requirements: If ultrafiltration combined with gel chromatography is used: large molecular immunogenic substances are removed by ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and then purified by Sephadex G-25 gel chromatography. If affinity chromatography is used: the enzymatic hydrolysate is passed through an affinity chromatography column (such as Protein A / G) to specifically adsorb immunogenic proteins, and non-immunogenic peptides are collected and purified by gel chromatography; If chemical modification is used: add an acylation reagent (such as succinic anhydride) to the purified peptide, react for 1-3 hours under pH 7.0-9.0 conditions to block the antigen epitope, and then remove the byproducts by gel chromatography; Finally, high-purity forest frog egg peptides without antigens were obtained.

[0015] S2. Preparation of microspheres: An improved single emulsion-solvent evaporation method was adopted. The frog egg peptide was dissolved in a water-soluble solvent, and the biodegradable polymer was dissolved in an organic solvent. The two were mixed to form a primary emulsion. Under stirring conditions, the primary emulsion was added dropwise to an external aqueous phase containing a surfactant to form a stable single emulsion. Then, the organic solvent was evaporated by means of reduced pressure evaporation or heating, and the biodegradable polymer was solidified to form microspheres.

[0016] S3. Enteric coating: The prepared microspheres are dispersed in an organic solution containing enteric material, and the microspheres are enteric coated by spray drying, fluidized bed coating or other methods to obtain enteric-protected frog egg peptide microspheres.

[0017] Specifically, in the enzymatic hydrolysis method, the amount of protease used is 0.5%-2% of the mass of the frog eggs.

[0018] Specifically, during the preparation of the microspheres, the stirring speed is 300-800 r / min and the stirring time is 2-5 h.

[0019] Specifically, during the enteric coating process, the inlet air temperature for spray drying is 110-130℃, and the outlet air temperature is 75-85℃.

[0020] Specifically, the water-soluble solvent is water, a dilute acid solution, or a dilute alkali solution, and the organic solvent is one or more of dichloromethane, chloroform, and ethyl acetate.

[0021] Specifically, in the preparation of microspheres, a surfactant is added to the external aqueous phase: "The external aqueous phase is an aqueous solution containing 0.5%-2% (w / v) surfactant (such as polyvinyl alcohol, Tween, Span).

[0022] Specifically, in enteric coating, the plasticizer and enteric material are co-soluble: "The enteric material (such as acrylic resin) and the plasticizer (such as 5%-10% triethyl citrate, accounting for the mass of the enteric material) are dissolved in an organic solvent (acetone / ethanol) to form a coating solution." Specifically, antioxidants can be added to the frog egg peptide solution: "When dissolving frog egg peptides, add 0.1%-0.5% (w / v) of antioxidants such as vitamin C to prevent peptide oxidation." Specifically, in the enteric coating step, the organic solution of the enteric material is 5%-10% (w / v) enteric material dissolved in acetone, ethanol or a mixture thereof (volume ratio 1:1), and 1%-5% plasticizer (such as polyethylene glycol) can be added to adjust the flexibility of the coating film.

[0023] The beneficial effects of this invention are: This invention significantly reduces the immunogenicity of the formulation by deantigenating frog ovpeptides (affinity chromatography to remove immunogenic proteins, chemical modification to block antigenic epitopes, and ultrafiltration combined with gel chromatography to remove macromolecular immunogenic substances), thereby reducing the risk of in vivo immune reactions and improving drug safety. The use of biodegradable polymers combined with enteric-coating materials ensures the stability of microspheres in the stomach, allowing for controlled drug release after entering the intestines. This solves the problem of acid degradation of frog ovpeptides in the stomach, significantly improving drug bioavailability. The improved single-emulsion-solvent evaporation method simplifies the preparation process, reduces complex steps, and lowers production costs and quality control difficulties. Simultaneously, optimized extraction processes and the use of controlled artificial breeding technology improve raw material utilization and achieve sustainable use of raw materials. The formulation exhibits stable drug release and high absorption efficiency, making it suitable for the enteric-coated formulation application of frog ovpeptides and other bioactive substances. Attached Figure Description

[0024] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] Figure 1 The flowchart illustrates the preparation method provided by this invention. Detailed Implementation

[0026] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0027] like Figure 1 As shown, the enteric-coated frog egg peptide microsphere formulation of the present invention comprises frog egg peptide, a biodegradable polymer, an enteric coating material, and optional additives. The biodegradable polymer is one or more of polylactic-coated glycolic acid copolymer (PLGA), polylactic acid (PLA), and chitosan. The enteric coating material is one or more of acrylic resin, hydroxypropyl methylcellulose phthalate (HPMCP), and cellulose acetate phthalate (CAP). Additives include, but are not limited to, antioxidants, plasticizers, and surfactants. The mass ratio of frog egg peptide to biodegradable polymer is 1:(2-10), and the amount of enteric coating material is 5%-20% of the total mass of the microspheres.

[0028] The preparation method of the enteric-coated frog egg peptide microsphere formulation includes the following steps: S1. Extraction and deantigenation of frog egg peptides: Wash fresh frog eggs, add phosphate buffer to homogenize, and enzymatically hydrolyze them (using papain, trypsin or pepsin, at a rate of 0.5%-2% of the frog egg mass) for 3-4 hours at a suitable temperature (37-38℃) and pH (2.0 or 7.0-8.0).

[0029] After enzymatic hydrolysis, centrifuge (8000 r / min, 30 min) and filter to remove impurities. Select the following treatment method according to the antigen removal requirements: Method ①: Ultrafiltration combined with gel chromatography Ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da removes large molecular weight immunogenic substances with a molecular weight greater than 3000 Da. The filtrate is then passed through a gel chromatography column (such as Sephadex G-25) and eluted with 0.1 mol / L sodium chloride solution. The elution peak containing frog ovpeptide is collected and freeze-dried.

[0030] Method 2: Affinity Chromatography The enzymatic hydrolysate is passed through an affinity chromatography column (such as a Protein A / G chromatography column) to specifically adsorb and remove immunogenic proteins (such as ovalbumin), and the unbound non-immunogenic peptide components are collected and then purified by gel chromatography (same as method ①).

[0031] Method 3: Chemical modification The purified frog egg peptide (after preliminary purification by ultrafiltration or gel chromatography) was chemically modified by adding an acylation reagent (such as succinic anhydride, with a peptide to reagent molar ratio of 1:5) and reacting at pH 7.0-9.0 for 1-3 hours to block the antigen epitope. After the reaction, unreacted reagents and byproducts were removed by gel chromatography and then freeze-dried.

[0032] Finally, high-purity forest frog egg peptides without antigens were obtained.

[0033] S2. Preparation of microspheres: An improved monoemulsion-solvent evaporation method was used, in which frog egg peptides were dissolved in a water-soluble solvent (water, dilute acid or dilute alkali solution), and biodegradable polymers (PLGA, PLA or chitosan) were dissolved in an organic solvent (dichloromethane, trichloromethane, etc.), and the two were mixed to form a primary emulsion. Under stirring conditions (300-800 r / min, 2-5 h), the primary emulsion was added dropwise to an external aqueous phase containing surfactants (polyvinyl alcohol, Tween, etc., concentration 0.5%-2% w / v) to form a stable monoemulsion. The organic solvent was evaporated by vacuum evaporation or heating, and the polymer solidified to form microspheres. The microspheres were collected by centrifugation, washed and dried.

[0034] S3. Enteric coating: Microspheres are dispersed in an organic solution (acetone, ethanol, etc.) containing enteric materials (acrylic resin, HPMCP, etc.), and 1%-5% plasticizer (triethyl citrate, etc.) is added. The target formulation is obtained by spray drying (inlet air temperature 110-130℃, outlet air temperature 75-85℃) or fluidized bed coating.

[0035] Example 1 Extraction and purification of *Rana lindana* ovpeptides: 100g of fresh *Rana lindana* eggs were washed three times with deionized water, and then 200mL of phosphate buffer (pH 7.4) was added. The mixture was homogenized for 10 min using a tissue homogenizer under ice bath conditions. 1g of papain was added to the homogenate, and the mixture was enzymatically hydrolyzed at 37℃ and pH 7.0 for 4 h. After hydrolysis, the mixture was centrifuged at 8000 rpm for 30 min, and the supernatant was collected and filtered through a 0.45μm microporous membrane. The filtrate was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the retentate was collected. The retentate was then purified by Sephadex G-25 gel chromatography, eluted with 0.1mol / L sodium chloride solution, and the elution peak containing *Rana lindana* ovpeptides was collected. The elution peak was then freeze-dried to obtain *Rana lindana* ovpeptide powder.

[0036] Preparation of microspheres: 500 mg of polylactic acid-glycolic acid copolymer (PLGA, LA:GA = 75:25) was dissolved in 5 mL of dichloromethane. 100 mg of the above-mentioned *Rana cristatum* ovpeptide powder was dissolved in 1 mL of distilled water. The *Rana cristatum* ovpeptide solution was slowly added dropwise to the PLGA dichloromethane solution. The mixture was ultrasonically emulsified for 3 min under ice bath conditions using an ultrasonic cell disruptor to form a primary emulsion. The primary emulsion was then slowly added dropwise to 100 mL of distilled water containing 1% (w / v) polyvinyl alcohol (PVA). The mixture was stirred at 25 °C and 500 r / min for 3 h to allow the dichloromethane to evaporate, forming microspheres. The microspheres were collected by centrifugation, washed three times with deionized water, and freeze-dried to obtain *Rana cristatum* ovpeptide microspheres.

[0037] Enteric coating: 100 mg of acrylic resin II was dissolved in 5 mL of acetone. 500 mg of the prepared *Rana cristatus* ovpeptide microspheres were dispersed in the acetone solution of the acrylic resin and stirred at 30 °C and 100 r / min for 1 h. The dispersion was then spray-dried using a spray dryer with an inlet air temperature of 120 °C and an outlet air temperature of 80 °C to obtain the *Rana cristatus* ovpeptide microsphere formulation with enteric coating protection.

[0038] Performance testing: The particle size of the microspheres was determined using a laser particle size analyzer, and the results showed an average particle size of (15.2±2.1) μm. The encapsulation efficiency of the microspheres was determined using high-performance liquid chromatography (HPLC), and the encapsulation efficiency was (85.6±3.2)%. The microsphere formulation was placed in artificial intestinal fluid, and the drug release rate was determined using dynamic dialysis. The results showed that the drug release was less than 10% within 2 hours, and the drug release was relatively stable within 6-12 hours, with a cumulative release of over 80%.

[0039] The immunogenicity of the microsphere formulation was detected by enzyme-linked immunosorbent assay (ELISA), and the results showed a weak immune response.

[0040] Example 2 Extraction and purification of *Rana lindana* ovum peptides: 150g of fresh *Rana lindana* eggs were washed and added to 300mL of phosphate buffer (pH 7.2). The mixture was homogenized for 15min using a high-speed disperser. 1.5g of trypsin was added, and the mixture was enzymatically hydrolyzed at 38℃ and pH 8.0 for 3.5h. Subsequent centrifugation, filtration, ultrafiltration, and gel chromatography purification steps were the same as in Example 1 to obtain *Rana lindana* ovum peptide powder.

[0041] Preparation of microspheres: 600 mg of polylactic acid (PLA) was dissolved in 6 mL of chloroform, and 120 mg of frog egg peptide powder was dissolved in 1.2 mL of distilled water. The mixture was then ultrasonically emulsified to form a primary emulsion. The primary emulsion was added dropwise to 120 mL of distilled water containing 1.2% (w / v) PVA, and the mixture was stirred for 4 h to allow the chloroform to evaporate. The microspheres were collected, washed, and dried.

[0042] Enteric coating: Weigh 120 mg of hydroxypropyl methylcellulose phthalate (HPMCP) and dissolve it in 6 mL of ethanol. Disperse the microspheres in the ethanol, stir to coat, and then spray dry. The inlet air temperature is 130 °C and the outlet air temperature is 85 °C.

[0043] Performance testing: The average particle size was (16.5±2.3) μm, the encapsulation rate was (84.8±3.5)%, the release rate in artificial intestinal fluid was less than 10% within 2 hours, and the cumulative release rate was more than 80% within 6-12 hours. Immunogenicity testing showed a weak immune response.

[0044] Example 3 Extraction and purification of frog egg peptides: Take 200g of fresh frog eggs, add 400mL of phosphate buffer solution with pH 7.0 to homogenize, add 2g of pepsin, and enzymatically hydrolyze at 37℃ and pH 2.0 for 4h. The subsequent purification steps are the same as before to obtain frog egg peptide powder.

[0045] Preparation of microspheres: Dissolve 700 mg chitosan in 1% acetic acid solution and 150 mg frog egg peptide powder in water. After mixing, add the mixture dropwise to 150 mL of distilled water containing 1.5% (w / v) PVA. Stir and crosslink to solidify to form microspheres. If the biodegradable polymer contains chitosan, add a crosslinking agent (such as 0.1%-1% glutaraldehyde aqueous solution) after the primary emulsion is added to the external aqueous phase. Stir and crosslink at 30-40℃ for 1-2 h. Then remove the crosslinking agent residue by centrifugation and washing, and wash and dry.

[0046] Enteric coating: 150 mg cellulose acetate phthalate (CAP) was dissolved in 7 mL of a mixed solvent of acetone and ethanol (volume ratio 1:1). After microsphere dispersion and coating, the microspheres were spray-dried at an inlet air temperature of 125°C and an outlet air temperature of 82°C.

[0047] Performance testing: average particle size (14.8±2.0) μm, encapsulation efficiency (86.2±3.0)%, release of artificial intestinal fluid within 2 hours less than 10%, cumulative release of more than 80% within 6-12 hours, and low immunogenicity.

[0048] Example 4 Extraction and purification of *Rana lindana* ovpeptides: 100g of fresh *Rana lindana* eggs were washed three times with deionized water, and then 200mL of phosphate buffer (pH 7.4) was added. The mixture was homogenized for 10 min using a tissue homogenizer under ice bath conditions. 1g of papain was added to the homogenate, and the mixture was enzymatically hydrolyzed at 37℃ and pH 7.0 for 4 h. After hydrolysis, the mixture was centrifuged at 8000 rpm for 30 min, and the supernatant was collected and filtered through a 0.45μm microporous membrane. The filtrate was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the retentate was collected. The retentate was then purified by Sephadex G-25 gel chromatography, eluted with 0.1mol / L sodium chloride solution, and the elution peak containing *Rana lindana* ovpeptides was collected. The elution peak was then freeze-dried to obtain *Rana lindana* ovpeptide powder.

[0049] Preparation of microspheres: 500 mg PLGA (LA:GA=50:50) was dissolved in 5 mL of dichloromethane, and 100 mg of frog ovum peptide powder was dissolved in 1 mL of distilled water containing 0.1% (w / v) Tween-80. After ultrasonic emulsification, the mixture was added dropwise to 100 mL of distilled water containing 1% (w / v) PVA and 0.05% (w / v) Span-80. The solvent was evaporated by stirring, and the microspheres were collected.

[0050] Enteric coating: 100 mg of acrylic resin II was dissolved in 5 mL of acetone. 500 mg of the prepared *Rana cristatus* ovpeptide microspheres were dispersed in the acetone solution of the acrylic resin and stirred at 30 °C and 100 r / min for 1 h. The dispersion was then spray-dried using a spray dryer with an inlet air temperature of 120 °C and an outlet air temperature of 80 °C to obtain the *Rana cristatus* ovpeptide microsphere formulation with enteric coating protection.

[0051] Performance testing: average particle size (15.8±2.2) μm, encapsulation efficiency (87.0±2.8)%, good release performance in artificial intestinal fluid, and low immunogenicity.

[0052] Example 5 Extraction and purification of *Rana lindana* ovum peptides: 150g of fresh *Rana lindana* eggs were washed and added to 300mL of phosphate buffer (pH 7.2). The mixture was homogenized for 15min using a high-speed disperser. 1.5g of trypsin was added, and the mixture was enzymatically hydrolyzed at 38℃ and pH 8.0 for 3.5h. Subsequent centrifugation, filtration, ultrafiltration, and gel chromatography purification steps were the same as in Example 1 to obtain *Rana lindana* ovum peptide powder.

[0053] Preparation of microspheres: 600 mg PLA was dissolved in 6 mL of chloroform, and 120 mg of frog ovum peptide powder was dissolved in 1.2 mL of distilled water containing 0.08% (w / v) Span-60. After emulsification, the mixture was added dropwise to 120 mL of distilled water containing 1.2% (w / v) PVA and 0.06% (w / v) Tween-20 and stirred to form microspheres.

[0054] Enteric coating: Weigh 120 mg of hydroxypropyl methylcellulose phthalate (HPMCP) and dissolve it in 6 mL of ethanol. Disperse the microspheres in the ethanol, stir to coat, and then spray dry. The inlet air temperature is 130 °C and the outlet air temperature is 85 °C.

[0055] Performance testing: average particle size (16.8±2.4) μm, encapsulation efficiency (85.0±3.3)%, stable release in artificial intestinal fluid, and low immunogenicity.

[0056] Example 6 Extraction and purification of frog egg peptides: Take 200g of fresh frog eggs, add 400mL of phosphate buffer solution with pH 7.0 to homogenize, add 2g of pepsin, and enzymatically hydrolyze at 37℃ and pH 2.0 for 4h. The subsequent purification steps are the same as before to obtain frog egg peptide powder.

[0057] Preparation of microspheres: 700 mg chitosan was dissolved in 1% acetic acid solution, 150 mg frog egg peptide powder was dissolved in distilled water containing 0.12% (w / v) polysorbate-85, and added dropwise to 150 mL of distilled water containing 1.5% (w / v) PVA and 0.08% (w / v) Span-40, and cross-linked and solidified into spheres.

[0058] Enteric coating: 150 mg cellulose acetate phthalate (CAP) was dissolved in 7 mL of a mixed solvent of acetone and ethanol (volume ratio 1:1). After microsphere dispersion and coating, the microspheres were spray-dried at an inlet air temperature of 125°C and an outlet air temperature of 82°C.

[0059] Performance testing: average particle size (14.5±1.9) μm, encapsulation efficiency (86.5±2.9)%, drug release in artificial intestinal fluid meets requirements, and immunogenicity is low.

[0060] Performance data comparison table of Examples 1-6:

[0061] Example 7 (Ultrafiltration + Gel Chromatography Antigen Removal Treatment) 1. Extraction and deantigenation of frog egg peptides: Take 200g of fresh frog eggs, wash them, add 400mL of pH7.4 phosphate buffer to homogenize, add 2g of trypsin, and enzymatically hydrolyze at 37℃ for 4h. The enzymatic hydrolysate was centrifuged at 8000 r / min for 30 min, and the supernatant was passed through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to remove large molecular weight immunogenic proteins. The ultrafiltration solution was passed through a Sephadex G25 gel chromatography column (column volume 100 mL, elution buffer 0.1 M NaCl), the elution peak was collected, and the solution was freeze-dried to obtain deantigenated forest frog ovpeptide (immunogenic protein residue <3%).

[0062] 2. Microsphere preparation and enteric coating: Weigh 800mg PLGA and dissolve it in 8mL dichloromethane; dissolve 100mg deantigenated frog ovpeptide in 1mL distilled water containing 0.1% vitamin C; and emulsify by sonication to form a primary emulsion. The primary emulsion was added dropwise to an external aqueous phase containing 1.5% PVA, the solvent was stirred to evaporate, the microspheres were collected and coated with a 5% hydroxypropyl methylcellulose phthalate ethanol solution, and spray-dried at an inlet air temperature of 125℃.

[0063] 3. Performance Testing: Immunogenicity test: After intraperitoneal injection of the preparation into mice, the serum titer of specific antibodies decreased by 75% compared with the untreated group (ELISA method).

[0064] Example 8 (Affinity chromatography for antigen removal) 1. Affinity chromatography removes immunogenic proteins: After enzymatic digestion and centrifugation according to the method in Example 1, the supernatant was passed through a pre-equilibrated Protein A affinity chromatography column (50 mL packing volume, 0.01 M PBS equilibration solution, pH 7.4). The unbound peptide fraction was eluted with 0.1M glycine-HCl buffer (pH 2.5), dialyzed, and then freeze-dried to obtain frog egg peptides with an immunogenic protein removal rate >90%.

[0065] 2. Microsphere preparation: Chitosan (dissolved in 1% acetic acid solution) and frog egg peptide (mass ratio 5:1) were mixed and added dropwise to an external aqueous phase containing 1% CaCl2. The mixture was cross-linked and solidified to form microspheres. The subsequent enteric coating steps were the same as in Example 3.

[0066] 3. Performance Testing: Encapsulation rate was 86.8%, and immunoassay showed no significant antibody production (Western blot method).

[0067] Example 9 (Chemical Modification and Antigen De-antigen Treatment) 1. Extraction and deantigenation of frog egg peptides: Take 100g of fresh frog eggs, and after enzymatic hydrolysis, centrifugation, and filtration according to the method in Example 1, pass the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da and collect the filtrate (containing small molecule peptides).

[0068] Succinic anhydride (acylation reagent, peptide to reagent molar ratio 1:5) was added to the purified *Rana cristatus* ovopeptide solution (concentration 1 mg / mL), and the reaction was carried out at room temperature for 2 h at pH 8.0 to block the antigenic epitopes of lysine residues on the peptide surface. After the reaction, unreacted reagents were removed by passing the solution through a Sephadex G-25 gel chromatography column (column volume 50 mL, elution buffer 0.1 M NaCl), and the elution peak was collected and freeze-dried to obtain the chemically modified deantigenated *Rana cristatus* ovopeptide (immunogenicity reduced by more than 70%, as detected by ELISA).

[0069] 2. Microsphere preparation and enteric coating: 600 mg of polylactic acid (PLA) was dissolved in 6 mL of chloroform. 120 mg of chemically modified frog ovum peptide was dissolved in 1.2 mL of distilled water containing 0.05% vitamin E. The mixture was then ultrasonically emulsified to form a primary emulsion. The primary emulsion was added dropwise to 120 mL of distilled water containing 1.2% (w / v) polyvinyl alcohol. The mixture was stirred for 4 hours to allow the chloroform to evaporate. The microspheres were collected, washed, and dried.

[0070] The enteric coating process is the same as in Example 2, using hydroxypropyl methylcellulose phthalate (HPMCP) for coating.

[0071] 3. Performance Testing: The average particle size was (16.0±2.5) μm, the encapsulation efficiency was (85.2±3.0)%, the release rate in artificial intestinal fluid within 2 hours was <10%, the cumulative release rate from 6 to 12 hours was >80%, and the immunogenicity test showed that the antibody production was reduced by 72% compared with the untreated group.

[0072] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A forest frog egg peptide microsphere formulation with enteric coating protection, characterized in that, include: Forest frog egg peptides treated with antigen removal, wherein the antigen removal treatment is any one or more of the following: ① Affinity chromatography removes immunogenic proteins; ② Chemical modification to block antigenic epitopes; ③Ultrafiltration combined with gel chromatography removes large molecular weight immunogenic substances with a molecular weight >3000 Da; The biodegradable polymer is one or more of polylactic acid-glycolic acid copolymer, polylactic acid, and chitosan; The enteric material is one or more of acrylic resin, hydroxypropyl methylcellulose phthalate, and cellulose acetate phthalate; Additives include one or more of antioxidants, plasticizers, and surfactants; The mass ratio of the frog egg peptide to the biodegradable polymer is 1:(2-10), and the amount of enteric material used is 5%-20% of the total mass of the microspheres.

2. The enteric-coated frog egg peptide microsphere formulation according to claim 1, characterized in that: The antioxidant is one or more of vitamin C, vitamin E, and propyl gallate.

3. The enteric-coated frog egg peptide microsphere formulation according to claim 1, characterized in that: The plasticizer is one or more of triethyl citrate, diethyl phthalate, and polyethylene glycol.

4. The enteric-coated frog egg peptide microsphere formulation according to claim 1, characterized in that: The surfactant is one or more of polyvinyl alcohol, Tween, and Span.

5. A method for preparing the enteric-coated frog egg peptide microsphere formulation according to claim 1, characterized in that, Includes the following steps: Step 1: Extraction and deantigenation of frog egg peptides: After enzymatic hydrolysis of fresh frog eggs, centrifugation and filtration are performed to remove impurities. The appropriate method for removing antigens is selected. ① Ultrafiltration: Large molecular immunogenic substances are removed by using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, followed by purification by gel chromatography; Or ② Affinity chromatography: The enzymatic hydrolysate is passed through an affinity chromatography column to remove immunogenic proteins, and the polypeptide components are collected and purified by gel chromatography; Or ③ ​​Chemical modification method: The purified peptide is subjected to acylation reaction to block the antigen epitope, and then the reaction product is removed by gel chromatography; Step 2, preparation of microspheres: Using an improved single emulsion-solvent evaporation method, frog egg peptides and biodegradable polymers were dissolved separately and mixed to form a primary emulsion, which was then added dropwise to an external aqueous phase containing surfactants. The organic solvent was evaporated and solidified to form microspheres. Step 3, enteric coating: The microspheres are dispersed in an organic solution of enteric material and coated by spray drying or fluidized bed to obtain the target formulation.

6. The method for preparing the enteric-coated frog egg peptide microsphere formulation according to claim 5, characterized in that: In the enzymatic hydrolysis method, the amount of protease used is 0.5%-2% of the mass of the frog eggs.

7. The method for preparing the enteric-coated frog egg peptide microsphere formulation according to claim 5, characterized in that: During the preparation of the microspheres, the stirring speed is 300-800 r / min and the stirring time is 2-5 h.

8. The method for preparing the enteric-coated frog egg peptide microsphere formulation according to claim 5, characterized in that: During the enteric coating process, the inlet air temperature for spray drying is 110-130℃, and the outlet air temperature is 75-85℃.

9. The method for preparing the enteric-coated frog egg peptide microsphere formulation according to claim 5, characterized in that: The water-soluble solvent is water, a dilute acid solution, or a dilute alkali solution, and the organic solvent is one or more of dichloromethane, trichloromethane, and ethyl acetate.

10. The method for preparing the enteric-coated frog egg peptide microsphere formulation according to claim 5, characterized in that: In the preparation of the microspheres, the surfactant is one or more of polyvinyl alcohol, Tween, and Span, which are added to the external aqueous phase at a concentration of 0.5%-2% w / v. When the frog egg peptide is dissolved in a water-soluble solvent, 0.1%-0.5% w / v of an antioxidant is added, wherein the antioxidant is one or more of vitamin C, vitamin E, and propyl gallate; The enteric material is added to an organic solution containing 1%-5% plasticizer by mass of the enteric material. The plasticizer is one or more of triethyl citrate, diethyl phthalate, and polyethylene glycol.

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