Ointment containing beta-cyclodextrin included egg yolk antibody as well as preparation method and application of ointment

Abstract

The invention provides an ointment containing a beta-cyclodextrin included egg yolk antibody and a preparation method thereof, and the ointment comprises the following raw materials by weight: 2%-5% of an emulsifier, 1.8%-2.9% of a thickener, 5%-10% of a humectant, 0.03%-0.1% of a pH regulator, 7%-20% of grease, 0.5%-0.9% of a preservative, 0.01%-0.5% of a metal ion chelating agent, and the balance of water. 0.2%-1% of a beta-cyclodextrin included egg yolk antibody, and the balance of water. The preparation method comprises the following steps: firstly, adding an emulsifier, grease and an oil-soluble thickening agent into a container in proportion, heating and stirring to prepare an oil-phase liquid, then adding purified water, a metal ion chelating agent, a humectant and a water-based thickening agent into another container, heating, stirring and uniformly mixing to prepare a water-phase liquid, further slowly pouring the oil-phase liquid into the water-phase liquid, and uniformly stirring to prepare the water-based oil-soluble thickening agent. The preparation method comprises the following steps: mixing the raw materials in a homogenizer, emulsifying in the homogenizer, and finally adding a preservative, a beta-cyclodextrin included egg yolk antibody and a pH regulator to prepare the final ointment, and the ointment can improve the stability of the egg yolk antibody and realize the slow-release effect of the egg yolk antibody.

Description

Technical Field

[0001] The invention relates to the field of biological preparations, in particular to a method for preparing an ointment by mixing beta-cyclodextrin with an egg yolk antibody, which can improve the stability and biological activity of the egg yolk antibody in an ointment matrix. Background Art

[0002] Yolk antibody (Immunoglobulin Y, IgY) is a specific antibody extracted from the egg yolk of poultry (such as chicken). IgY is the most important antibody in the avian immune system. It has similar functions to mammalian IgG, but is unique in structure and biochemical properties. Different antigens, such as various inflammatory factors and inactivated viruses and bacteria, can be injected into the body of chickens to obtain antibodies with different functions. Due to its simple production, low cost and wide source, IgY has received widespread attention in the fields of animal immunity, medical diagnosis and biological products in recent years.

[0003] However, there are also certain challenges in the application of egg yolk antibodies. First, egg yolk antibodies have a certain degree of environmental sensitivity and are easily degraded under high temperature, acidic environment and enzymatic conditions, which limits their application in complex topical preparations; secondly, egg yolk antibodies are large molecules with poor solubility, and are prone to precipitation or aggregation in many preparations, affecting their stability and biological activity in the preparations. In addition, in topical preparations such as ointments, IgY is easily inactivated due to environmental changes (such as pH, humidity), resulting in a decrease in effect; or due to the uneven distribution of antibodies in topical preparations, the activity of IgY cannot be effectively exerted.

[0004] β-cyclodextrin, as a commonly used inclusion material, has the characteristics of a hollow molecular structure, and can include target molecules in its cavity, thereby improving the stability and solubility of the target molecules. β-cyclodextrin inclusion technology has been widely used in drug delivery and protection of active substances. For example, by including small molecule drugs in β-cyclodextrin, their solubility, stability and bioavailability can be improved. However, there has been no systematic study combining β-cyclodextrin inclusion technology with egg yolk antibodies to develop a stable preparation method suitable for topical ointments.

[0005] The present invention aims to solve the following technical problems existing in the prior art in the application of egg yolk antibodies in external ointments:

[0006] 1. The yolk antibody molecules are large and poorly soluble, and are prone to precipitation or aggregation.

[0007] Yolk antibody molecules are large and have poor solubility, and are prone to precipitation or aggregation in many preparations. This not only affects their stability in the preparations, but may also lead to reduced antibody activity, limiting their application effect in preparations.

[0008] 2. The stability of yolk antibodies in complex environments is insufficient

[0009] In the prior art, when preparing topical ointments, yolk antibodies (IgY) are easily affected by changes in the external environment (such as pH, temperature and humidity), resulting in a decrease in antibody activity or even complete inactivation, which cannot meet the needs of long-term storage and actual use of topical preparations.

[0010] 3. Uneven distribution of antibodies in ointment preparations

[0011] In the existing preparation methods of protein drug ointments, the distribution of antibodies is often difficult to control, resulting in uneven concentration distribution at the target site, affecting the treatment effect. Summary of the invention

[0012] In view of the existing technical problems, the present invention provides an ointment containing β-cyclodextrin-encapsulated egg yolk antibodies. The egg yolk antibodies are encapsulated in β-cyclodextrin and then combined with other ointment raw materials to form an ointment containing egg yolk antibodies. The activity of the egg yolk antibodies in the ointment can be maintained, the antibodies are evenly distributed in the ointment, and the concentration is evenly distributed at the target site, thereby promoting skin repair and care.

[0013] To achieve the above objectives, the specific technical solutions of the present invention are as follows:

[0014] The present invention provides an ointment containing beta-cyclodextrin-encapsulated egg yolk antibody, which contains the following raw materials in percentage by weight: 2%-5% emulsifier, 1.8%-2.9% thickener, 5%-10% moisturizer, 0.03%-0.1% pH regulator, 7%-20% oil, 0.5%-0.9% preservative, 0.01%-0.5% metal ion chelating agent, 0.2%-1% beta-cyclodextrin-encapsulated egg yolk antibody, and the balance is made up of water.

[0015] The emulsifier is selected from PEG-100 glyceryl stearate, glyceryl stearate, and cetearyl glucoside, cetearyl olive oil ester, sorbitan olive oil ester, PEG-20 methyl glucose sesquistearate, methyl glucose sesquistearate, steareth-21, steareth-2, and a combination of two or more thereof, preferably in a ratio of 2% to 4%;

[0016] The thickener is one or more of carbomer 980, xanthan gum, cetearyl alcohol or hydroxyethyl cellulose, preferably carbomer 980, and the preferred ratio is 2%-2.5%;

[0017] The moisturizing agent is selected from one or more of glycerol, propylene glycol, butylene glycol, 1.2-pentanediol, 1.2-hexanediol, recombinant humanized type III collagen or sodium hyaluronate, preferably glycerol, preferably in a proportion of 6%-9%;

[0018] The pH regulator is selected from one of sodium hydroxide, arginine and triethanolamine, preferably in a ratio of 0.05% to 0.08%;

[0019] The oil is selected from shea butter, vaseline, white oil, silicone oil, oat kernel oil, vitamin E, any three or more of the combination, preferably in a ratio of 10%-20%;

[0020] The preservative is selected from one or a combination of two or more of methylparaben, sodium benzoate, and phenoxyethanol, preferably in a ratio of 0.5% to 0.6%;

[0021] The metal ion chelating agent is EDTA-2Na, preferably in a ratio of 0.05% to 0.3%;

[0022] The beta-cyclodextrin-encapsulated egg yolk antibody is prepared by the following steps:

[0023] 1. Immunogen

[0024] The immunogens are various inflammatory factors (IL31, TNF-α, IL-6, etc.), enzyme proteins (COX-1, SOD, etc.), pathogen-related proteins (viral surface proteins, bacterial toxins, etc.), inactivated or attenuated pathogenic microorganisms (such as inactivated HIV virus, attenuated measles virus, inactivated Salmonella, attenuated Mycobacterium leprae, etc.), immunogenic regions of natural proteins (such as antigenic epitopes of viral capsid proteins and antigenic epitopes of bacterial exotoxins, etc.), or polysaccharides and glycoproteins (such as pneumococcal capsular polysaccharides, Vibrio cholerae lipopolysaccharide (LPS), human glycoprotein antigens (such as hCG)) and other antigens.

[0025] 2. Immunization of laying hens

[0026] Select 120-day-old laying hens, which are ordinary laying hens, and fully mix the selected antigen with Freund's complete adjuvant in a volume ratio of 1:1 until the emulsion is stable. The laying hens are immunized and boosted by subcutaneous injection, with 0.1ml-1ml injected subcutaneously into the back or pectoral muscle of each hen. After the initial immunization, the chickens are given booster injections every 1 to 3 weeks, for a total of 2 booster injections. Starting from the 30th to 40th day after the initial immunization, eggs are collected every day and stored below 4°C.

[0027] 3. Isolation and purification of IgY

[0028] The collected eggs are disinfected with 75% alcohol, and the yolk and egg white are separated with an egg yolk separator under aseptic conditions. The egg yolk is taken for use, and the egg yolk is diluted with 5-10 times the volume of pure water of the egg yolk and stirred. The pH value is adjusted to 4.5-7.0 using 0.5M hydrochloric acid solution, and the mixture is left standing at 4°C for 4-12 hours. The mixture is centrifuged at 4°C and 12000g for 10 minutes, and the supernatant is collected, which contains the precipitate of crude egg yolk antibody. Saturated ammonium sulfate is added to the supernatant to make its saturation 50%, and the mixture is left standing at 4°C overnight. The mixture is centrifuged at 4°C and 12000g for 10 minutes, and the supernatant is discarded. The precipitate is resuspended and dissolved with 10% of the volume of egg yolk. The dissolved precipitate is dialyzed with PBS (the dialysate volume is 50 times the volume of the liquid in the dialysis bag) for more than 20 hours, and the dialysate is replaced once every 6-8 hours to obtain an egg yolk antibody solution. The titer of egg yolk antibody is detected using indirect ELISA.

[0029] 4. Inclusion of yolk antibodies

[0030] β-cyclodextrin, a hydrophilic wall material with a mass of 0.1 to 4 times that of the egg yolk antibody, is mixed with the egg yolk antibody, the inner diameter (A°) of the β-cyclodextrin molecular cavity is 6.0-6.5, the mixed solution is emulsified and sheared at a speed of 9000r / min at 35-40°C for 1.5-2h to obtain antibody colostrum, and then transferred to an ultra-high pressure emulsification homogenizer, homogenized 2-3 times at 200-420Mpa, each time for 3-5 minutes, after homogenization, the solution is freeze-dried to obtain β-cyclodextrin-encapsulated egg yolk antibody powder, and stored at -20°C for use;

[0031] The preferred weight proportion of the beta-cyclodextrin-embedded egg yolk antibody in the ointment is 0.2%-0.8%.

[0032] The ointment preparation method comprises the following steps:

[0033] 1. Preparation of oil phase: Add emulsifier, oil and oil-soluble thickener into a clean container in proportion, place the container in a water bath at 75℃-80℃ and heat, stirring while heating at a speed of 200-400rpm until completely dissolved to form a uniform oil phase liquid;

[0034] 2. Preparation of water phase: Add purified water to another container, add moisturizer and thickener in proportion, heat to 75℃-80℃, keep stirring at 200-400rpm until completely dissolved to form a uniform solution;

[0035] 3. Emulsification: at a constant temperature (80°C-85°C), slowly pour the oil phase into the water phase and homogenize with a homogenizer at a speed of 6000-10000rpm, preferably 6000-10000rpm, for about 5-10 minutes to fully emulsify the oil and water phases to form a uniform emulsion;

[0036] 4. Cooling and stabilization: After emulsification is completed, continue stirring until the temperature is below 45°C, add preservatives, and continue stirring evenly with a mixer at a speed of 200-400rpm / min, preferably 200-300rpm. When the temperature drops to about 40°C, add the egg yolk antibody encapsulated by β-cyclodextrin, continue stirring evenly at a constant speed, and add a pH adjuster as needed to adjust the pH value to between 4.5 and 6.5.

[0037] The ointment containing beta-cyclodextrin-encapsulated egg yolk antibody prepared by the invention can be used to relieve skin itching and dryness.

[0038] The present invention utilizes beta-cyclodextrin to include egg yolk antibodies, and successfully prepares an external ointment with good stability and antibody activity by optimizing the inclusion process and the combination process with the ointment matrix to meet the use requirements of the ointment.

[0039] Beneficial effects of technical solutions

[0040] 1. Improve the stability of yolk antibodies

[0041] Anti-environmental damage: Yolk antibodies are easily affected by external factors (such as temperature, humidity, light, etc.) during storage and use and lose their activity. The present invention utilizes the inclusion properties of β-cyclodextrin to encapsulate the yolk antibody at the molecular level to form a stable complex, effectively reducing its contact with the external environment, thereby significantly improving the stability and shelf life of the antibody.

[0042] Prevent protein degradation: The inclusion effect of β-cyclodextrin can reduce the exposure of key sites during protein degradation and further prolong the activity of yolk antibodies.

[0043] 2. Achieve sustained-release effect and enhance the efficacy of ointment

[0044] The complex formed by β-cyclodextrin inclusion has good sustained-release properties and can control the release rate of egg yolk antibodies.

[0045] When applied in an ointment, it can continuously release egg yolk antibodies, ensuring long-term maintenance of activity, reducing the frequency of use, and improving the effect of use and user compliance.

[0046] 3. Improve the performance and usage experience of ointment

[0047] Texture optimization: β-cyclodextrin and egg yolk antibody complex are mixed with the ointment base, which not only ensures uniform distribution of ingredients, but also gives the ointment better touch and ductility.

[0048] Enhanced absorbability: The ointment prepared by the present invention improves the skin penetration performance of the active ingredient through the carrier effect of β-cyclodextrin, making the egg yolk antibody more easily absorbed by the target site, improving the overall efficacy of the product, and improving the unpleasant odor of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The solutions are 1% unencapsulated egg yolk antibody aqueous solution, 1% β-cyclodextrin aqueous solution and 1% β-cyclodextrin encapsulated egg yolk antibody aqueous solution. DETAILED DESCRIPTION

[0050] 1. Preparation of the yolk antibody used in the examples of the present invention:

[0051] (1) Immunogen selection

[0052] Interleukin 31 (IL31) was purchased from Univ Biotech.

[0053] (2) Immunization of laying hens

[0054] 120-day-old laying hens were selected, and the laying hens were ordinary laying hens. IL31 and Freund's adjuvant were fully mixed in a volume ratio of 1:1 until the emulsion was stable. The laying hens were immunized and boosted by subcutaneous injection. 0.5 ml of the mixed emulsion of IL31 and Freund's adjuvant was injected subcutaneously on the back of each hen. After the initial immunization, the chickens were injected with the same dose of booster shots every two weeks, for a total of 2 booster shots. Starting from the 35th day after the initial immunization, eggs were collected every day and stored below 4°C.

[0055] (3) Isolation, purification and inclusion of IgY

[0056] Take the collected 20 eggs and disinfect them with 75% alcohol. Under sterile conditions, separate the yolk and egg white with an egg yolk separator, mix the egg yolk and set aside, dilute the egg yolk with 9 times the volume of pure water of the egg yolk and stir, use 0.5M hydrochloric acid solution to adjust the pH value to 5.5, and let it stand at 4°C for 6 hours. Centrifuge the mixture at 4°C and 12000g for 10 minutes, collect the supernatant, and obtain a precipitate containing crude IgY. Add saturated ammonium sulfate to the supernatant to make its saturation 50%, let it stand at 4°C overnight, centrifuge the mixture at 4°C and 12000g for 10 minutes, discard the supernatant, and resuspend and dissolve the precipitate with 10% of the volume of egg yolk in pure water. The dissolved precipitate was dialyzed with PBS (the dialysate volume was 50 times the volume of the liquid in the dialysis bag) for 25 hours, and the dialysate was replaced every 6 hours to obtain the IgY solution. The titer of anti-IL31 egg yolk antibody was detected using indirect ELISA (the kit was purchased from Shanghai Keshun Biotechnology Co., Ltd.): IL31 was diluted to 40 ng / well with coating solution, added to the ELISA plate, 100 μL per well, incubated at 37°C for 2 h, the coating solution was discarded, and the plate was incubated with Wash 3 times with PBS containing Tween-20, 1 min each time; add 200 μL of PBS containing Tween-20 to each well. Bovine serum albumin PBS solution, blocked at 4°C overnight, washed as above; the samples to be tested were Bovine serum albumin PBS solution was diluted 1:500 and then 2-fold in 8 gradients and added to the ELISA plate, 100 μL per well, incubated at 37°C for 2 h, and washed 5 times as above; enzyme-labeled goat anti-chicken secondary antibody was diluted 1:20000 with PBS solution and added to the ELISA plate, 100 μL per well, incubated at 37°C for 1 h, and washed 5 times as above; 100 μL TMB substrate colorimetric solution was added to each well, incubated at 37°C in the dark for 15 min, and then 100 μL stop solution was added to each well to terminate the color development reaction. The OD value (OD450) at a wavelength of 450 nm was read using an ELISA instrument, and the concentration of the yolk antibody was measured to be 100 mg / ml.

[0057] 2. Preparation of β-cyclodextrin-encapsulated egg yolk antibody

[0058] 21.25g of the hydrophilic wall material β-cyclodextrin was mixed with 500ml of the egg yolk antibody solution prepared above, the inner diameter (A°) of the β-cyclodextrin molecular cavity was 6.0-6.5, the mixed solution was emulsified and sheared at 9000r / min for 1.5h at 35°C to obtain antibody colostrum, and then transferred to an ultra-high pressure emulsification homogenizer, homogenized 3 times at 320Mpa, each time for 5 minutes, after the homogenization was completed, the solution was freeze-dried to obtain β-cyclodextrin-encapsulated egg yolk antibody powder, and stored at -20°C for later use;

[0059] 3. Confirm the inclusion is successful

[0060] 1% mass concentration of unencapsulated egg yolk antibody aqueous solution, 1% mass concentration of β-cyclodextrin aqueous solution and 1% mass concentration of β-cyclodextrin encapsulated egg yolk antibody aqueous solution were prepared respectively. The results showed that the 1% unencapsulated egg yolk antibody aqueous solution was slightly turbid, while the 1% β-cyclodextrin aqueous solution and the 1% β-cyclodextrin encapsulated egg yolk antibody aqueous solution were clear and transparent, proving that the egg yolk antibody had been encapsulated successfully. Figure 1 shown.

[0061] Embodiment 1:

[0062] The raw material ratio of the ointment containing β-cyclodextrin-encapsulated egg yolk antibody is calculated by weight percentage:

[0063] Emulsifier: Steareth-211%, Steareth-21%

[0064] Thickener: Xanthan gum 0.2%, Carbomer 9800.1%, Cetearyl alcohol 1.5%

[0065] Humectants: Glycerin 1%, Propylene Glycol 4%, Butylene Glycol 5%

[0066] pH adjuster: Sodium hydroxide 0.03%

[0067] Oils and fats: Shea butter 3%, white oil 1%, petrolatum 3%

[0068] Preservatives: Sodium benzoate 0.5%, phenoxyethanol 0.4%

[0069] Metal ion chelating agent: EDTA-2Na 0.01%

[0070] Yolk antibody: Use 0.2% yolk antibody encapsulated with β-cyclodextrin

[0071] Water: Make up to 100% with purified water.

[0072] Production steps:

[0073] 1. Preparation of oil phase: Add steareth-21, steareth-2, shea butter, white oil, vaseline and cetearyl alcohol into a clean container in proportion, heat and stir in a water bath at 80°C until completely dissolved to form a uniform oil phase liquid.

[0074] 2. Preparation of aqueous phase: Add purified water to another container, add Carbopol 980 first, stir evenly, then add metal ion chelator EDTA-2Na, then add glycerol, propylene glycol, butylene glycol and xanthan gum in sequence, heat to 80°C and stir until completely dissolved.

[0075] 3. Emulsification: Slowly pour the oil phase into the water phase, use a homogenizer at a speed of 6000 rpm, homogenize at 85°C for about 5 minutes, stir evenly to form an emulsion.

[0076] 4. Cooling and stabilization: After emulsification, use a stirrer to cool down to 45°C at a speed of 200 rpm, add sodium benzoate and phenoxyethanol, add sodium hydroxide in batches to adjust the pH value of the formula to 5.8, and continue to stir until uniform. When the temperature drops to 40°C, add β-cyclodextrin-encapsulated egg yolk antibody, keep the speed unchanged, stir evenly, and obtain the final ointment.

[0077] Embodiment 2:

[0078] The raw material ratio of the ointment containing β-cyclodextrin-encapsulated egg yolk antibody is calculated by weight percentage:

[0079] Emulsifier: Cetearyl Glucoside 1%, PEG-20 Methyl Glucose Sesquistearate 2.5%, Methyl Glucose Sesquistearate 1.5%

[0080] Thickener: Hydroxyethylcellulose 0.2%, Carbomer 9800.1%, Cetearyl Alcohol 2.6%

[0081] Moisturizer: Sodium Hyaluronate 0.05%, Propylene Glycol 4.95%

[0082] pH adjuster: triethanolamine 0.1%

[0083] Oils and fats: Silicone oil 6%, Vitamin E 0.25%, Oat kernel oil 4%, Shea butter 3.75%, White oil 2%, Vaseline 4%

[0084] Preservative: Phenoxyethanol 0.5%

[0085] Metal ion chelating agent: EDTA-2Na 0.5%

[0086] Water: Make up to 100% with purified water.

[0087] Production steps:

[0088] 1. Preparation of oil phase: Add cetearyl glucoside, PEG-20 methyl glucose sesquistearate, methyl glucose sesquistearate and silicone oil, vitamin E, oat kernel oil, shea butter, white oil, vaseline and cetearyl alcohol into a clean container in proportion, heat and stir in a 80°C water bath until completely dissolved to form a uniform oil phase liquid.

[0089] 2. Preparation of aqueous phase: Add purified water to another container, first add hydroxyethyl cellulose and carbomer 980, then add ion chelator EDTA-2Na, then add sodium hyaluronate and propylene glycol in sequence, heat to 80°C and stir until completely dissolved.

[0090] 3. Emulsification: Slowly pour the oil phase into the water phase, use a homogenizer at a speed of 10,000 rpm, homogenize at 85°C for about 5 minutes, stir evenly to form an emulsion.

[0091] 4. Cooling and stabilization: After emulsification, use a stirrer to cool down to 45°C at a speed of 400 rpm, add triethanolamine to adjust the pH value of the formula to 5.3, and then add phenoxyethanol and continue to stir until uniform. When the temperature drops to 40°C, add the yolk antibody encapsulated by β-cyclodextrin, keep the speed unchanged, stir evenly, and obtain the final ointment.

[0092] Embodiment 3:

[0093] The raw material ratio of the ointment containing β-cyclodextrin-encapsulated egg yolk antibody is as follows:

[0094] Emulsifier: PEG-100 Glyceryl Stearate 2.5%, Glyceryl Stearate 1.2%

[0095] Thickener: Carbomer 9800.4%, Cetearyl Alcohol 2%

[0096] Humectant: Glycerin 4%, 1.2-pentanediol 3%, 1.2-hexanediol 0.99%, recombinant humanized type III collagen 0.01%

[0097] pH adjuster: Sodium hydroxide 0.08%

[0098] Grease: white oil 3%, vaseline 2%, silicone oil 5%

[0099] Preservatives: Phenoxyethanol 0.4%, Methylparaben 0.2%

[0100] Metal ion chelating agent: EDTA-2Na 0.1%

[0101] Yolk antibody: Use 0.4% yolk antibody encapsulated with β-cyclodextrin

[0102] Water: Pure water to 100%

[0103] Production steps:

[0104] 1. Preparation of oil phase: Add PEG-100 glyceryl stearate, glyceryl stearate and white oil, vaseline, silicone oil and cetearyl alcohol in a clean container in proportion, heat and stir in an 80°C water bath until completely dissolved to form a uniform oil phase liquid.

[0105] 2. Preparation of aqueous phase: Add purified water to another container, add Carbopol 980 first, stir evenly, then add metal ion chelator EDTA-2Na, then add glycerol, 1.2-pentanediol, and 1.2-hexanediol in sequence, heat to 80°C and stir until completely dissolved.

[0106] 3. Emulsification: Slowly pour the oil phase into the water phase, use a homogenizer at a speed of 8000 rpm, homogenize at 85°C for about 5 minutes, stir evenly to form an emulsion.

[0107] 4. Cooling and stabilization: After emulsification, use a stirrer to cool down to 45°C at a speed of 300 rpm, add sodium hydroxide, adjust the pH value of the product to 5.8, then add phenoxyethanol and methylparaben and continue to stir until uniform. When the temperature drops to 40°C, add β-cyclodextrin-encapsulated egg yolk antibody and recombinant humanized type III collagen, keep the speed unchanged, stir evenly, and obtain the final ointment.

[0108] Comparative Example 1 (addition of unencapsulated egg yolk antibody):

[0109] The raw material ratio of the ointment containing yolk antibodies:

[0110] Emulsifier: PEG-100 Glyceryl Stearate 2.5%, Glyceryl Stearate 1.2%

[0111] Thickener: Carbomer 9800.4%, Cetearyl Alcohol 2%

[0112] Humectant: Glycerin 4%, 1.2-pentanediol 3%, 1.2-hexanediol 0.99%, recombinant humanized type III collagen 0.01%

[0113] pH adjuster: Sodium hydroxide 0.08%

[0114] Grease: white oil 3%, vaseline 2%, silicone oil 5%

[0115] Preservatives: Phenoxyethanol 0.4%, Methylparaben 0.2%

[0116] Metal ion chelating agent: EDTA-2Na 0.1%

[0117] Yolk antibody: Use 0.4% yolk antibody not encapsulated by β-cyclodextrin

[0118] Water: Make up to 100% with purified water.

[0119] Production steps:

[0120] 1. Preparation of oil phase: Add PEG-100 glyceryl stearate, glyceryl stearate and white oil, vaseline, silicone oil and cetearyl alcohol in a clean container in proportion, heat and stir in an 80°C water bath until completely dissolved to form a uniform oil phase liquid.

[0121] 2. Preparation of aqueous phase: Add purified water to another container, add Carbopol 980 first, stir evenly, then add metal ion chelator EDTA-2Na, then add glycerol, 1.2-pentanediol, 1.2-hexanediol and methylparaben in sequence, heat to 80°C and stir until completely dissolved.

[0122] 3. Emulsification: Slowly pour the oil phase into the water phase, use a homogenizer at a speed of 8000 rpm, homogenize at 85°C for about 5 minutes, stir evenly to form an emulsion.

[0123] 4. Cooling and stabilization: After emulsification, use a stirrer to cool down to 45°C at a speed of 300 rpm, add sodium hydroxide, adjust the pH value of the product to 5.8, then add phenoxyethanol and continue to stir until uniform. When the temperature drops to 40°C, add the egg yolk antibody that is not encapsulated by β-cyclodextrin and recombinant humanized type III collagen, keep the speed unchanged, and stir evenly.

[0124] Comparative Example 2 (without adding yolk antibody):

[0125] Emulsifier: PEG-100 Glyceryl Stearate 2.5%, Glyceryl Stearate 1.2%

[0126] Thickener: Carbomer 9800.4%, Cetearyl Alcohol 2%

[0127] Humectant: Glycerin 4%, 1.2-pentanediol 3%, 1.2-hexanediol 0.99%, recombinant humanized type III collagen 0.01%

[0128] pH adjuster: Sodium hydroxide 0.08%

[0129] Grease: white oil 3%, vaseline 2%, silicone oil 5%

[0130] Preservatives: Phenoxyethanol 0.4%, Methylparaben 0.2%

[0131] Metal ion chelating agent: EDTA-2Na 0.1%

[0132] Water: Pure water to 100%

[0133] Production steps:

[0134] 1. Preparation of oil phase: Add PEG-100 glyceryl stearate, glyceryl stearate and white oil, vaseline, silicone oil and cetearyl alcohol in a clean container in proportion, heat and stir in an 80°C water bath until completely dissolved to form a uniform oil phase liquid.

[0135] 2. Preparation of aqueous phase: Add purified water to another container, add Carbopol 980 first, stir evenly, then add metal ion chelator EDTA-2Na, then add glycerol, 1.2-pentanediol, 1.2-hexanediol and methylparaben in sequence, heat to 80°C and stir until completely dissolved.

[0136] 3. Emulsification: Slowly pour the oil phase into the water phase, use a homogenizer at a speed of 8000 rpm, homogenize at 85°C for about 5 minutes, stir evenly to form an emulsion.

[0137] 4. Cooling and stabilization: After emulsification, use a stirrer at 300 rpm to cool down to below 45°C, add sodium hydroxide, adjust the pH value of the product to 5.6, then add phenoxyethanol and continue to stir until uniform. When the temperature drops to 40°C, add recombinant humanized type III collagen, keep the speed unchanged, and stir evenly.

[0138] Application Examples

[0139] Skin sensitization of the ointment prepared in Test Example 3:

[0140] 1. Skin sensitization test (human skin patch test)

[0141] According to the method of Chapter VII.2 of the "Technical Specification for Safety of Cosmetics" (2015 edition), a human skin patch test was conducted to detect the potential possibility of the ointment of the present invention causing adverse reactions on human skin:

[0142] Materials and methods:

[0143] Test substance: the ointment of Example 3 of the present invention.

[0144] Negative control: blank control (no substance is placed).

[0145] Subjects: A total of 30 subjects, including 2 males and 28 females, aged 23 to 54 years old, with an average age of 37±10 years old, met the voluntary inclusion criteria.

[0146] Patch test method: A patch tester (purchased from Beijing Baiyi Yida Technology Development Co., Ltd.) was used. 0.025 g of the test substance was placed in the patch tester by closed patch test method. The test substance was applied to the forearm of the subject with low-sensitivity tape. The test substance was removed after 24 hours. The skin reaction was observed at 0.5, 24, and 48 hours after removal. The results were recorded according to the skin reaction grading standard of the "Safety Technical Specifications for Cosmetics" (2015 edition):

[0147] Table 1 Skin reaction grading standards for skin occlusive patch test

[0148]

[0149] Test results:

[0150] Table 2 Summary of cosmetic human skin patch test results

[0151]

[0152] The results of the human skin patch test showed that no adverse skin reactions occurred in 30 people. This proves that the ointment prepared by the present invention is safe for the skin. Since the raw materials for preparing the ointment in Comparative Example 1 are the same as those in Example 3 except that the egg yolk antibody is not included by β-cyclodextrin, the ointment prepared in Comparative Example 1 is also safe for the skin.

[0153] The skin sensitization of the ointments prepared in Examples 1 and 2 and Comparative Example 2 was tested by the same method as above. The experimental results showed that no adverse skin reactions occurred in the subjects.

[0154] 2. Antibody activity test

[0155] 2.1 Temperature stability test:

[0156] The samples prepared on the same day in Example 3 and Comparative Example 1 were stored at 48° C. for 7 days, 14 days and 30 days, respectively. Samples were taken at each time point, and the relative activity of the yolk antibody in each group of samples was determined using enzyme-linked immunosorbent assay (ELISA).

[0157] 2.2 Humidity stability test

[0158] The samples prepared on the same day in Example 3 and Comparative Example 1 were placed in a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 80%. The test was conducted for 7 days, 14 days and 30 days. Samples were taken at each time point and the relative activity in each group of samples was determined using ELISA.

[0159] 2.3 Light stability test

[0160] The samples prepared on the same day as Example 3 and Comparative Example 1 were exposed to ultraviolet light (UVA) to simulate 4 hours of direct sunlight per day and placed in a dark place for the rest of the time. The test was conducted for 7 and 14 days, and the relative activity of yolk antibodies in the light-exposed group and the completely dark-protected group was detected using ELISA.

[0161] Calculate the retention rate of antibody activity:

[0162] Activity retention rate = (activity after storage / initial activity) × 100%

[0163] Experimental results: see Table 3

[0164] Table 3 Antibody activity retention results

[0165]

[0166] The experimental results show that the activity retention rate of β-cyclodextrin-encapsulated egg yolk antibodies under high temperature, humidity and light conditions is significantly higher than that of unencapsulated antibodies. The increase in antibody activity retention after inclusion increases with storage time. β-cyclodextrin inclusion technology can effectively improve the stability of egg yolk antibodies, especially under extreme environmental conditions and during long-term storage, and can significantly reduce activity loss.

[0167] 3. Clinical efficacy trial

[0168] To evaluate the efficacy and safety of an ointment containing β-cyclodextrin-encapsulated egg yolk antibodies and compare it with an ointment without egg yolk antibodies or an ointment without added egg yolk antibodies, in order to verify the advantages of the encapsulated antibodies.

[0169] 60 subjects with confirmed eczema and stable condition, without serious infection or other chronic skin diseases, aged between 18 and 55 years old, who had not received other immunomodulatory treatments in the past month were randomly selected and divided into 3 groups, 20 people in each group. The first group used the ointment prepared in Example 3, the second group used the ointment prepared in Comparative Example 1, and the third group used the ointment prepared in Comparative Example 2.

[0170] Usage: The three groups used ointment prepared from the same batch, once in the morning and once in the evening. After cleaning the skin, evenly apply the designated product, with the edge of the application exceeding the edge of the eczema by 1 cm and the amount of application covering the eczema. Apply it continuously for 4 weeks, and follow up every week to record the changes.

[0171] Main evaluation indicators:

[0172] Improvement in eczema severity: using the EASI score (range 0-72, the lower the score, the milder the condition).

[0173] Scoring criteria:

[0174] 1. Assessment areas of EASI scoring

[0175] The body is divided into four regions, each occupying a different proportion of the total area:

[0176] Head / Neck: 10% (0.1)

[0177] Upper Limbs: 20% (0.2)

[0178] Trunk (including genitals): 30% (0.3)

[0179] Lower Limbs: 40% (0.4)

[0180] Skin lesion area score

[0181] 2. Scoring based on the percentage of eczema lesions covered:

[0182] 0 points: no skin lesions (0%)

[0183] 1 point: skin lesion coverage < 10%

[0184] 2 points: Lesions cover 10-29%

[0185] 3 points: Lesions cover 30-49%

[0186] 4 points: Lesions cover 50-69%

[0187] 5 points: skin lesions cover 70-89%

[0188] 6 points: 90-100% skin coverage

[0189] 3. In each area, rate the severity of the following 4 characteristics of the eczematous lesions:

[0190] Erythema: 0 (none) to 3 (severe)

[0191] Edema / Induration or Papulation: 0 (none) to 3 (severe)

[0192] Excoriation: 0 (none) to 3 (severe)

[0193] Lichenification: 0 (none) to 3 (severe)

[0194] Each score ranges from 0 to 3 and is summed to give an overall severity score for that area (0 to 12 points).

[0195] 4. EASI score calculation formula

[0196] The score for each region is calculated using the following formula:

[0197] EASI = ∑ region (area score × total severity score × region weight coefficient),

[0198] Right now:

[0199] EASI=(AH×SH×0.1)+(AU×SU×0.2)+(AT×ST×0.3)+(AL×SL×0.4)

[0200] in:

[0201] A is the skin lesion area score

[0202] S is the sum of the severity scores (erythema + infiltration / thickening + scratching / breakage + lichenification)

[0203] H, U, T, L represent head and neck, upper limbs, trunk and lower limbs respectively.

[0204] 6. Interpretation of EASI scores

[0205] 0 points: no eczema

[0206] 1-7 points: Mild eczema

[0207] 8-21 points: Moderate eczema

[0208] 22-50 points: severe eczema

[0209] >50 points: Very severe eczema

[0210] Itch relief: Patients' subjective itching score (0-10 points).

[0211] Scoring criteria:

[0212] 0 points: no itching

[0213] The patient had no itching sensation at all.

[0214] 1-3 points: Mild itching

[0215] The itching sensation occurs occasionally, but it hardly interferes with daily life and is easily ignored.

[0216] 1 point: Almost no itching, only noticeable when paying attention.

[0217] 2 points: Slight itching, but it disappears quickly.

[0218] 3 points: Itching is somewhat obvious, but can be ignored and does not require special treatment.

[0219] 4-6 points: moderate itching

[0220] The itching sensation lasts for a long time, and patients often feel itching, which begins to interfere with their daily life.

[0221] 4 points: Itching occasionally interrupts concentration but does not affect sleep.

[0222] 5 points: The itching is quite obvious, and daily life is often disturbed. Scratching may be needed to relieve it.

[0223] 6 points: The itching sensation is intense and frequent, daily activities are significantly affected, and scratching becomes uncontrollable.

[0224] 7-8 points: severe itching

[0225] The itching sensation is very obvious, and the patient's daily life, mood and sleep are significantly disturbed.

[0226] 7 points: The itching seriously affects daily activities and is unbearable, requiring frequent scratching or drug intervention.

[0227] 8 points: The itching sensation is close to the limit, the patient feels irritable or painful, and it is difficult to concentrate on other things.

[0228] 9-10 points: Extremely severe itching

[0229] The itching is unbearable, persistent and severe, seriously affecting the quality of life, and patients may experience emotional breakdown or depression.

[0230] 9 points: The itching causes intense pain and makes it difficult to concentrate or sleep.

[0231] 10 points: The itching reaches the limit that the patient can tolerate and is completely unable to lead a normal life or rest.

[0232] Rating interpretation:

[0233] 0-3 points: no or mild itching, with basically no need for improvement.

[0234] 4-6 points: Active intervention is required to improve symptoms.

[0235] 7 points and above: indicates severe itching.

[0236] A decrease of 3 points or more is considered an improvement.

[0237] Treatment satisfaction: scored by patient questionnaire (1-5 points).

[0238] Scoring criteria:

[0239] 1 point: Very dissatisfied

[0240] The users do not recognize the improvement at all, there is no improvement, and the product has not achieved any of the expected goals.

[0241] 2 points: Unsatisfied,

[0242] Users believe that the product is not effective, with limited improvement and almost no noticeable changes, and are disappointed with the results.

[0243] 3 points: General (neutral)

[0244] Users believe that the effect of use has improved to some extent, but has not yet reached the expected goal. Users feel slight relief, but are still cautious about further use.

[0245] 4 points: Satisfactory

[0246] Users generally recognize the effects of use, which have achieved the main expected goals and greatly improved their quality of life.

[0247] 5 points: Very satisfied

[0248] Users highly recognize the effects of use, and the overall skin condition and experience far exceed expectations.

[0249] Incidence of adverse reactions: Record skin redness, itching, allergies and other reactions.

[0250] Detection time point:

[0251] Day 0 (baseline), day 7, day 14, day 28 after application of the ointment.

[0252] Experimental results: see Table 4

[0253] Table 4 Clinical effect test results

[0254]

[0255] Experimental conclusion:

[0256] (1) Improvement in eczema severity (EASI score):

[0257] The EASI score of Example 3 decreased from 40±10 at baseline to 1±4 on day 28, with a significant improvement of about 97.5%. The improvement was fast, with the score halved on day 7, showing an excellent therapeutic effect;

[0258] The EASI score of Comparative Example 1 decreased from 42±12 at baseline to 15±7 on day 28, a decrease of 64.3%. Although there was a significant improvement, the degree and speed of the reduction were inferior to those of Example 3;

[0259] The EASI score of control group 2 was almost unchanged compared with the baseline score of 44±14, and was still 44±14 on day 28, showing no effective improvement.

[0260] (2) Itch score:

[0261] The pruritus score of Example 3 decreased from 6±1 at baseline to 0.5±0.5 on day 28, with a reduction of 91.7%. The rapid decrease in the pruritus score reflects the significant relief of pruritus by the use of the ointment;

[0262] The pruritus score of Comparative Example 1 decreased from 6.5±1.5 at the baseline to 2±1 on the 28th day, with a reduction of 69.2%, which was inferior to that of Example 3;

[0263] The pruritus score of control example 2 only decreased from 6±1 at baseline to 5±3 on day 28, and the change was not significant.

[0264] (3) Patient satisfaction:

[0265] The satisfaction rate of Example 3 on the 7th day was 4±0.5, and on the 28th day was 5±0, showing a trend of high satisfaction. The users had the highest recognition of the overall experience.

[0266] The satisfaction rate of comparative example 1 on the 7th day was 3±1, and on the 28th day it was 4±1, which was moderate and had some improvement, but the subjective feeling of the user was not optimal.

[0267] The satisfaction score of Example 2 remained at 2±0.5, with almost no change, reflecting dissatisfaction with the use.

[0268] The results showed that the EASI score of Example 3 decreased rapidly, the severity of eczema improved significantly, the itching score was almost completely relieved, the patient satisfaction reached the highest, and the incidence of adverse reactions was the lowest. It was significantly better than the other two groups. The severity of eczema and itching in Comparative Example 1 were improved, but the magnitude and speed were not as good as the first group, the satisfaction was low, and the incidence of adverse reactions was high. Comparative Example 2 had almost no effect, and there was no significant change in the severity of eczema and itching scores, and the patient satisfaction was low. Therefore, the beta-cyclodextrin inclusion yolk antibody product is significantly better than the non-inclusion yolk antibody in improving eczema symptoms, relieving itching and restoring skin barrier function, and has good safety and higher patient compliance.

[0269] The above experimental results prove that the use of β-cyclodextrin-encapsulated egg yolk antibodies improves the stability and biological activity of egg yolk antibodies in the ointment. The ointment made with β-cyclodextrin-encapsulated egg yolk antibodies has better temperature, humidity and light stability, the antibody is evenly distributed, and has a better use effect.

Claims

1. An ointment containing β-cyclodextrin-encapsulated egg yolk antibody, characterized in that: The invention comprises the following raw materials in percentage by weight: 2%-5% of emulsifier, 1.8%-2.9% of thickener, 5%-10% of moisturizer, 0.03%-0.1% of pH regulator, 7%-20% of oil, 0.5%-0.9% of preservative, 0.01%-0.5% of metal ion chelator, 0.2%-1% of beta-cyclodextrin-encapsulated egg yolk antibody, and the balance is water.

2. The ointment containing β-cyclodextrin-encapsulated egg yolk antibody according to claim 1, characterized in that: The preparation process of β-cyclodextrin-encapsulated egg yolk antibody is as follows: the hydrophilic wall material β-cyclodextrin and the egg yolk antibody are mixed in a mass ratio of 0.1-4:1, the mixed solution is emulsified and sheared at a speed of 9000r / min at 35-40°C for 1.5-2h to obtain antibody colostrum, and then transferred to an ultra-high pressure emulsification homogenizer, homogenized 2-3 times at 200-420Mpa, each time for 3-5 minutes. After the homogenization is completed, the β-cyclodextrin-encapsulated egg yolk antibody is obtained, and the inner diameter of the β-cyclodextrin molecular cavity (A°) is 6.0-6.

5.

3. The ointment containing β-cyclodextrin-encapsulated egg yolk antibody according to claim 1, characterized in that: The emulsifier is selected from two or more of PEG-100 glyceryl stearate, glyceryl stearate, cetearyl glucoside, cetearyl olivate, sorbitan olivate, PEG-20 methyl glucose sesquistearate, methyl glucose sesquistearate, steareth-21, and steareth-2, and the preferred ratio is 2%-4%.

4. The ointment containing β-cyclodextrin-encapsulated egg yolk antibody according to claim 1, characterized in that: The thickener is one or more of Carbomer 980, xanthan gum, cetearyl alcohol or hydroxyethyl cellulose, preferably Carbomer 980, and the preferred ratio is 2%-2.5%.

5. The ointment containing β-cyclodextrin-encapsulated egg yolk antibody according to claim 1, characterized in that: The moisturizing agent is selected from one or more of glycerol, propylene glycol, butylene glycol, 1.2-pentanediol, 1.2-hexanediol, recombinant humanized type III collagen or sodium hyaluronate, preferably glycerol, and the preferred ratio is 6%-9%.

6. The ointment containing β-cyclodextrin-encapsulated egg yolk antibody according to claim 1, characterized in that: The pH regulator is selected from one of sodium hydroxide, arginine and triethanolamine, preferably in a ratio of 0.05% to 0.08%; The oil is selected from shea butter, vaseline, white oil, silicone oil, oat kernel oil, vitamin E, any three or more of the combination, preferably in a ratio of 10%-20%; The preservative is selected from one or a combination of two or more of methylparaben, sodium benzoate, and phenoxyethanol, preferably in a ratio of 0.5% to 0.6%; The metal ion chelating agent is EDTA-2Na, and the preferred ratio is 0.05%-0.3%.

7. A method for preparing an ointment containing β-cyclodextrin-encapsulated egg yolk antibody according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Preparation of oil phase: Add emulsifier, oil and oil-soluble thickener in a clean container according to proportion, place the container in a water bath at 75°C-80°C and heat, stirring at a speed of 200-400 rpm, until the emulsifier is completely dissolved to form a uniform oil phase liquid; (2) Preparation of aqueous phase: Add purified water to another container, add metal ion chelating agent, humectant and thickener in proportion, heat to 75°C-80°C, keep stirring at a speed of 200-400 rpm until completely dissolved to form a uniform solution; (3) Emulsification: at a constant temperature of 80°C-85°C, slowly pour the oil phase into the water phase and homogenize with a homogenizer at a speed of 6000-10000 rpm, preferably 6000-10000 rpm, for 5-10 minutes to fully emulsify the oil and water phases to form a uniform emulsion; (4) Cooling and stabilization: After emulsification is completed, continue stirring until the temperature is below 45°C, add preservatives, and continue stirring evenly using a stirrer at a speed of 200-400 rpm, preferably 200-300 rpm. When the temperature drops to 35-40°C, add β-cyclodextrin-encapsulated egg yolk antibodies, continue stirring evenly, add a pH adjuster, and adjust the pH value to between 4.5 and 6.5 to obtain an ointment containing β-cyclodextrin-encapsulated egg yolk antibodies.

8. Use of the ointment containing β-cyclodextrin-encapsulated egg yolk antibody according to claim 1 for relieving skin itching and dryness.

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