Pineapple peptide ointment and preparation method thereof

Abstract

The application discloses a kind of pineapple peptide ointment and preparation method thereof, the raw materials of pineapple peptide ointment include pineapple peptide, thymus protein peptide, sheep placenta extract, bromelain, vitamin E, arbutin, humectant, thickening agent, penetrant and purified water;Wherein, according to the weight fraction, its proportion is as follows: 8-10% pineapple peptide, 2-4% thymus protein peptide, 4-6% sheep placenta extract, 3-5% bromelain, 2-4% vitamin E, 2-4% arbutin, 0.1-0.2% humectant, 0.5-1.0% thickening agent, 0.8-1.0% penetrant, the balance is purified water.The application provides a kind of pineapple peptide ointment capable of promoting scar repair, inhibiting scar proliferation, obtains scar repair composition with the effects of reducing pigment deposition, flattening scar thickness, softening scar texture and inhibiting scar proliferation, and the components contain a large amount of small molecular protein peptides, easy to be absorbed and utilized by organism.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and relates to ointments, particularly to a pineapple peptide ointment that can promote scar repair and inhibit scar hyperplasia, and its preparation method. Background Technology

[0002] Scars are localized symptoms that affect both appearance and function, resulting from damage to the skin's soft tissue caused by physical, biological, chemical, or other factors. This damage is often too severe for the skin to fully repair itself, leading to replacement by fibrous tissue. Scars cause immense physical and psychological suffering, especially those resulting from burns, scalds, or severe trauma. The proliferative phase of scarring can be excruciating for years, while the subsequent atrophic phase can leave patients disfigured and functionally impaired, leading to significant psychological and emotional trauma. Furthermore, in today's high-standard-of-living environment, people have high standards for their appearance and strive for perfection. Therefore, acne scars from puberty, stretch marks, or scars from everyday bumps and scrapes can all significantly impact one's quality of life.

[0003] Pineapple, a famous tropical fruit, is rich in aldoses, organic acids, proteins, fiber, and various essential vitamins and trace elements. It promotes digestion, cleanses the intestines, and quenches thirst, making it highly nutritious. Currently, the most common pineapple extract on the market is bromelain, which is used as a meat tenderizer, beer additive, and other food additives. In the medical field, it is also used to treat edema, inflammation, and wound healing, demonstrating its wide range of applications. Pineapple peptides are small-molecule plant polypeptides produced from pineapple fruit and stems using modern technologies such as enzymatic hydrolysis, membrane separation, and ultrafiltration. Currently, research reports on pineapple peptides are relatively limited.

[0004] Topical preparations for treating burns include both traditional Chinese medicine (TCM) and Western medicine preparations. Western medicine preparations are typically made from antibacterial substances, such as antibiotic ointments (e.g., tetracycline ointment, oxytetracycline ointment, chloramphenicol ointment, erythromycin ointment, ciprofloxacin ointment, norfloxacin ointment), and silver-containing ointments (e.g., silver sulfadiazine ointment, silver nitrate cream). These products are highly irritating to the skin, easily causing allergies and drug resistance, and have significant toxic side effects. TCM preparations are usually compound ointments, such as moist burn ointment, burn ointment, and burn analgesic ointment. They are generally oil-based products requiring high-temperature melting; some creams and water-soluble base ointments generally require the addition of preservatives to prevent mold growth. Furthermore, TCM ointments contain many raw materials, and the extraction of some TCM active ingredients is complex. For example, a certain burn ointment contains more than 30 kinds of TCM herbs, its extraction process is very complex, raw material costs are high, and the product cost is expensive. In addition, most commercially available ointments are only suitable for superficial (first-degree) burns, with little repair effect and obvious scarring after repair. Summary of the Invention

[0005] To address the aforementioned problems, the present invention first provides a pineapple peptide ointment, thereby obtaining a scar repair composition with effects such as reducing pigmentation, smoothing scar thickness, softening scar texture, and inhibiting scar hyperplasia.

[0006] The present invention further provides a method for preparing the above-mentioned pineapple peptide ointment.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A pineapple peptide ointment, wherein the raw materials of the pineapple peptide ointment include pineapple peptide, thymosin peptide, sheep placenta extract, bromelain, vitamin E, arbutin, moisturizer, thickener, penetrant, and purified water; wherein, by weight fraction, the proportions are: 8-10% pineapple peptide, 2-4% thymosin peptide, 4-6% sheep placenta extract, 3-5% bromelain, 2-4% vitamin E, 2-4% arbutin, 0.1-0.2% moisturizer, 0.5-1.0% thickener, 0.8-1.0% penetrant, and the balance being purified water.

[0009] As a preferred embodiment of the present invention, the moisturizer is selected from one of sodium hyaluronate, glycerol, butylene glycol, propylene glycol, sorbitol, xylitol, polyethylene glycol, polyglycerol, sodium lactate, pyrrolidone carboxylic acid, or urea.

[0010] Most preferably, the moisturizer in this invention is sodium hyaluronate.

[0011] As a preferred embodiment of the present invention, the thickener is selected from sodium carboxymethyl cellulose, propylene glycol alginate, polyoxyethylene, sodium polyacrylate, casein, or polyvinylpyrrolidone.

[0012] Most preferably, the thickener in this invention is sodium carboxymethyl cellulose.

[0013] As a preferred embodiment of the present invention, the penetrant is selected from one of azone, isosorbide dimethyl ether, thiazolinone, inositol or tetrahydropenetrose.

[0014] Most preferably, the penetrant in this invention is azone.

[0015] As a preferred embodiment of the present invention, the method for preparing the pineapple peptide is as follows:

[0016] 1) Take fresh pineapple fruit and stem, wash and drain them, then crush them with a grinder, then homogenize them in a homogenizer at 2-8℃ and treat them with ultrasound to obtain a homogenate. After removing the fiber residue by coarse filtration with a filter cloth, take the filtrate.

[0017] 2) Adjust the pH of the filtrate to 4.5-5.5 with sodium hydroxide solution, heat it in a water bath to 45-55℃, add 0.1%-0.2% of cellulase by weight of the slurry, keep it warm for 1-2 hours, and after the reaction is complete, heat it in a water bath to 75-85℃ and keep it warm for 10-20 minutes.

[0018] 3) After cooling to 50-60℃ at room temperature, adjust the pH to 6.0-7.0 with sodium hydroxide solution, add 0.2%-0.3% of compound flavor protease by weight of the slurry, keep warm for 2-4 hours, and after the reaction is completed, heat in a water bath to 75-80℃ and keep warm for 10-15 minutes.

[0019] 4) The enzymatic hydrolysate is concentrated by ultrafiltration using a series of 20K / 10K / 5KDa ultrafiltration membranes;

[0020] 5) The concentrated solution is placed in a sample tray and pre-cooled in a refrigerator for 30 minutes. Then, it is placed in a vacuum freeze dryer and freeze-dried according to the preset freeze-drying curve. The freeze-drying curve is to first freeze (temperature: about -35 to -45℃, pressure: about 13Pa) into a solid state, then sublimate (temperature: about -25℃ to -35℃, pressure: about 5Pa), and finally dry (35 to 45℃) to obtain pineapple peptide.

[0021] As a preferred embodiment of the present invention, the cellulase used in step 2) has an enzyme activity of 100,000 u / g; the compound flavor protease used in step 3) has an enzyme activity of 50,000 u / g.

[0022] As a preferred embodiment of the present invention, the method for preparing the thymocyte protein peptide is as follows:

[0023] a) Take fresh, healthy calf thymus and wash it several times with 0.9% physiological saline; cut it into small pieces with a grinder, and then homogenize it in a homogenizer at 2-8℃ to obtain a homogenate.

[0024] b) After freezing the homogenate in a freezer at -18°C, take it out and heat it in a water bath at 40°C to dissolve it. After it is completely dissolved, put it back into the freezer at -18°C and freeze it. Repeat this freeze-thaw cycle at least 3 times.

[0025] c) Adjust the pH of the above freeze-thawed solution to 2.0-3.0 with hydrochloric acid solution, heat it in a water bath to 35-45°C, add 0.4%-0.6% pepsin by weight of the slurry, and keep it warm for 2-4 hours;

[0026] d) After the reaction is complete, heat the mixture in a water bath to 75-85°C and keep it at that temperature for 10-20 minutes. Then, concentrate the mixture by ultrafiltration using a series of 20K / 10K / 5KDa ultrafiltration membranes.

[0027] e) The concentrate is placed in a sample tray and pre-cooled in a refrigerator for 30 minutes. Then, it is placed in a vacuum freeze dryer and freeze-dried according to the preset freeze-drying curve. The freeze-drying curve is to first freeze (temperature: about -30 to -40℃, pressure: about 13 Pa) into a solid state, then sublimate (temperature: about -35℃ to -45℃, pressure: about 5 Pa), and finally dry (35 to 45℃) to obtain thymocyte peptide.

[0028] As a preferred embodiment of the present invention, the pepsin used in step c) has an enzyme activity of 12,000 u / mg.

[0029] This invention also provides a method for preparing the above-mentioned pineapple peptide ointment, comprising the following steps:

[0030] S1. Preparation of the base: According to the formula, the thickener and humectant are dissolved in water to form a gel-like state. After being mixed evenly, the mixture is sterilized in a steam sterilizer to serve as the ointment base.

[0031] S2. Under sterile conditions, pineapple peptide, thymosin peptide, sheep placental extract and bromelain were dissolved in water in sequence to prepare a peptide solution.

[0032] S3. Pretreatment of other excipients: Under sterile conditions, arbutin and the penetrant are dissolved separately in purified water and ethanol, and then mixed to obtain an excipient solution;

[0033] S4. Preparation of ointment: Under sterile conditions, the peptide solution obtained in s2, the excipient solution obtained in s3, and vitamin E are added sequentially to the ointment base obtained in s1. The contents are weighed and calibrated, stirred and mixed, and sterilized in a steam sterilizer for 10-20 minutes. After cooling, the ointment is filled to obtain pineapple peptide ointment.

[0034] In a preferred embodiment of the present invention, in S1, the temperature of steam sterilization is 100°C; in S4, the temperature of steam sterilization is 80-85°C.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1) This invention provides a pineapple peptide ointment that can promote scar repair and inhibit scar hyperplasia, resulting in a scar repair composition with the effects of reducing pigmentation, smoothing scar thickness, softening scar texture and inhibiting scar hyperplasia. The composition contains a large number of small molecule protein peptides, which are easily absorbed and utilized by the body.

[0037] 2) This invention uses pineapple as raw material and obtains pineapple peptides through biological enzymatic hydrolysis, ultrafiltration and other methods. Compared with traditional peptide water extraction, acid-base extraction or adsorption extraction methods, it has the characteristics of high recovery rate, good purity, good biological activity, high purity and rich content of effective substances.

[0038] 3) The use of the pineapple peptide ointment of the present invention can effectively improve the physical and psychological trauma of a large number of people suffering from scars and increase their social self-confidence.

[0039] 4) The preparation process of the pineapple peptide ointment of the present invention is simple and easy to operate, without the introduction of any pollutants, and the production process has low energy consumption, so the product is highly operable for industrialization.

[0040] 5) The product of this invention is in the form of an ointment, which is suitable for scars on almost any part of the body, especially irregular scars.

[0041] 6) The pineapple peptide ointment of this invention uses a water-soluble matrix, which maintains the stability of each active ingredient while having the characteristics of rapid drug release and no irritation to the skin and mucous membranes. Detailed Implementation

[0042] To facilitate understanding of the technical means, creative features, objectives, and effects of this invention, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0043] In this invention, the pineapple peptide and thymosin peptide used are self-made products of the applicant company, while the sheep placenta extract, bromelain, vitamin E, arbutin, sodium carboxymethyl cellulose, sodium hyaluronate and azone are commercially available products.

[0044] Preparation of pineapple peptides:

[0045] 1) Take fresh pineapple fruit and stem, wash and drain them, then crush them with a grinder, then homogenize them in a homogenizer at 2-8℃ and treat them with ultrasound to obtain a homogenate. After removing the fiber residue by coarse filtration with a filter cloth, take the filtrate.

[0046] 2) Adjust the pH of the filtrate to 4.5-5.5 with sodium hydroxide solution, heat it in a water bath to 45-55℃, add 0.1%-0.2% of cellulase by weight of the slurry (enzyme activity: about 100,000 u / g), keep it warm for 1-2 hours, and after the reaction is complete, heat it in a water bath to 75-85℃ and keep it warm for 10-20 minutes.

[0047] 3) After cooling to 50-60℃ at room temperature, adjust the pH to 6.0-7.0 with sodium hydroxide solution, add 0.2%-0.3% of compound flavor protease (enzyme activity: about 50,000 u / g) by weight of slurry, keep warm for 2-4 hours, and after the reaction is completed, heat in a water bath to 75-80℃ and keep warm for 10-15 minutes.

[0048] 4) The enzymatic hydrolysate is concentrated by ultrafiltration using a series of 20K / 10K / 5KDa ultrafiltration membranes;

[0049] 5) The concentrated solution is placed in a sample tray and pre-cooled in a refrigerator for 30 minutes. Then, it is placed in a vacuum freeze dryer and freeze-dried according to the preset freeze-drying curve. The freeze-drying curve is to first freeze (temperature: about -35 to -45℃, pressure: about 13Pa) into a solid state, then sublimate (temperature: about -25℃ to -35℃, pressure: about 5Pa), and finally dry (35 to 45℃) to obtain pineapple peptide.

[0050] Preparation of thymocyte protein peptides:

[0051] a) Take fresh, healthy calf thymus (15 days old, yellow cattle), wash it several times with 0.9% physiological saline; cut it into pieces with a grinder, and then homogenize it in a homogenizer at 2-8℃ to obtain a homogenate.

[0052] b) After freezing the homogenate in a freezer at -18°C, take it out and heat it in a water bath at about 40°C to dissolve it. After it is completely dissolved, put it back into the freezer at -18°C and freeze it. Repeat this freeze-thaw cycle at least 3 times.

[0053] c) Adjust the pH of the above freeze-thawed solution to about 2.0-3.0 with hydrochloric acid solution, heat it in a water bath to 35-45°C, add 0.4%-0.6% of pepsin by weight (enzyme activity: about 12,000 u / mg), and keep it warm for 2-4 hours;

[0054] d) After the reaction is complete, heat the mixture in a water bath to 75-85°C and keep it at that temperature for 10-20 minutes. Then, concentrate the mixture by ultrafiltration using a series of 20K / 10K / 5KDa ultrafiltration membranes.

[0055] e) The concentrate is placed in a sample tray and pre-cooled in a refrigerator for 30 minutes. Then, it is placed in a vacuum freeze dryer and freeze-dried according to the preset freeze-drying curve. The freeze-drying curve is to first freeze (temperature: about -30 to -40℃, pressure: about 13 Pa) into a solid state, then sublimate (temperature: about -35℃ to -45℃, pressure: about 5 Pa), and finally dry (35 to 45℃) to obtain thymocyte peptides.

[0056] Example 1

[0057] Preparation of Pineapple Peptide Ointment Sample 1

[0058] (1) Preparation of the matrix: ① Add 150 mL of purified water to a 250 mL beaker and place it under an automatic rotary stirrer. Weigh out 5 g of sodium carboxymethyl cellulose and slowly and evenly sprinkle it into the beaker while stirring at 300 rpm (to ensure that the sodium carboxymethyl cellulose is evenly dispersed in the water). After the addition is complete, adjust the stirring speed to about 500 rpm and stir for 10 h. ② Add 200 mL of purified water to a 250 mL beaker and heat it to 70 °C in a water bath. Weigh out 1 g of sodium hyaluronate and slowly and evenly sprinkle it into the beaker while stirring (to ensure that the sodium hyaluronate is evenly dispersed in the water). Continue stirring for 20 min while keeping the temperature warm. After there are no obvious particles in the solution, transfer it to an automatic rotary stirrer (speed: about 500 rpm) and continue stirring for 1 h. ③ After mixing the materials in ① and ② evenly, sterilize them in a 100 °C autoclave for 10 min to obtain the ointment matrix.

[0059] (2) Preparation of peptide solution: Under sterile conditions, add 200mL of purified water to a 250mL beaker, weigh out 80g of pineapple peptide, 20g of thymosin peptide, 40g of sheep placental extract and 30g of bromelain, add them in sequence and stir to dissolve, to obtain a peptide mixed solution.

[0060] (3) Pretreatment of other excipients: Under sterile conditions, take 20g of arbutin and dissolve it in 40mL of purified water at about 45℃. Then, take another 8g of azone and dissolve it in 15mL of ethanol. Mix the two together and let them stand for later use.

[0061] (4) Preparation of ointment: Under sterile conditions, the peptide solution from step (2), the excipient solution from step (3), and 20g of vitamin E were added to the prepared matrix in sequence, and the weight was adjusted to 1000g with purified water. After stirring for 30 minutes to ensure complete mixing of the materials, the mixture was placed in a high-pressure steam sterilizer and sterilized at 80°C for 10 minutes. After cooling, the mixture was dispensed into PE bottles sterilized by ultraviolet light, 10g / bottle, for a total of 95 tubes of pineapple peptide scar ointment.

[0062] Example 2

[0063] Preparation of Pineapple Peptide Ointment Sample 2

[0064] (1) Preparation of the matrix: ① Add 150 mL of purified water to a 250 mL beaker and place it under an automatic rotary stirrer. Weigh out 7.5 g of sodium carboxymethyl cellulose and slowly and evenly sprinkle it into the beaker while stirring at 300 rpm (to ensure that the sodium carboxymethyl cellulose is evenly dispersed in the water). After the addition is complete, adjust the stirring speed to about 500 rpm and stir for 12.5 h. ② Add 200 mL of purified water to a 250 mL beaker and heat it to 70 °C in a water bath. Weigh out 1.5 g of sodium hyaluronate and slowly and evenly sprinkle it into the beaker while stirring (to ensure that the sodium hyaluronate is evenly dispersed in the water). Continue stirring for 20 min while keeping the temperature warm. After there are no obvious particles in the solution, transfer it to an automatic rotary stirrer (speed: about 500 rpm) and continue stirring for 1.5 h. ③ After mixing the materials in ① and ② evenly, sterilize them in a 100 °C autoclave for 10 min to obtain the ointment matrix.

[0065] (2) Preparation of peptide solution: Under sterile conditions, add 200mL of purified water to a 250mL beaker, weigh out 90g of pineapple peptide, 30g of thymosin peptide, 50g of sheep placental extract and 40g of bromelain, add them in sequence and stir to dissolve, to obtain a peptide mixed solution.

[0066] (3) Pretreatment of other excipients: Under sterile conditions, take 30g of arbutin and dissolve it in 40mL of purified water at about 45℃. Then, take another 9g of azone and dissolve it in 15mL of ethanol. Mix the two together and let them stand for later use.

[0067] (4) Preparation of ointment: Under sterile conditions, the peptide solution from step (2), the excipient solution from step (3), and 30g of vitamin E were added to the prepared matrix in sequence, and the weight was adjusted to 1000g with purified water. After stirring for 45min to ensure complete mixing of the materials, the mixture was placed in a high-pressure steam sterilizer and sterilized at 80℃ for 15min. After cooling, the mixture was dispensed into PE bottles sterilized by ultraviolet light, 10g / bottle, for a total of 94 tubes of pineapple peptide scar ointment.

[0068] Example 3

[0069] Preparation of Pineapple Peptide Ointment Sample 3

[0070] (1) Preparation of the matrix: ① Add 150 mL of purified water to a 250 mL beaker and place it under an automatic rotary stirrer. Weigh out 10 g of sodium carboxymethyl cellulose and slowly and evenly sprinkle it into the beaker while stirring at 300 rpm (to ensure that the sodium carboxymethyl cellulose is evenly dispersed in the water). After the addition is complete, adjust the stirring speed to about 500 rpm and stir for 15 h. ② Add 200 mL of purified water to a 250 mL beaker and heat it to 70 °C in a water bath. Weigh out 2 g of sodium hyaluronate and slowly and evenly sprinkle it into the beaker while stirring (to ensure that the sodium hyaluronate is evenly dispersed in the water). Continue stirring for 20 min while keeping the temperature warm. After there are no obvious particles in the solution, transfer it to an automatic rotary stirrer (speed: about 500 rpm) and continue stirring for 2 h. ③ After mixing the materials in ① and ② evenly, sterilize them in a 100 °C autoclave for 10 min to obtain the ointment matrix.

[0071] (2) Preparation of peptide solution: Under sterile conditions, add 200mL of purified water to a 250mL beaker, weigh 100g of pineapple peptide, 40g of thymosin peptide, 60g of sheep placental extract and 50g of bromelain, add them in sequence and stir to dissolve, to obtain a peptide mixed solution.

[0072] (3) Pretreatment of other excipients: Under sterile conditions, take 40g of arbutin and dissolve it in 40mL of purified water at about 45℃. Then, take another 10g of azone and dissolve it in 15mL of ethanol. Mix the two together and let them stand for later use.

[0073] (4) Preparation of ointment: Under sterile conditions, the peptide solution from step (2), the excipient solution from step (3), and 40g of vitamin E were added to the prepared matrix in sequence, and purified water was used to weigh up to 1000g. After stirring for 60min, the materials were completely mixed and then placed in a high-pressure steam sterilizer and sterilized at 85℃ for 20min. After cooling, the ointment was dispensed into PE bottles sterilized by ultraviolet light, 10g / bottle, for a total of 91 tubes of pineapple peptide scar ointment.

[0074] Example 4

[0075] Preparation of Pineapple Peptide Ointment Sample 4

[0076] (1) Preparation of the matrix: ① Add 150 mL of purified water to a 250 mL beaker and place it under an automatic rotary stirrer. Weigh out 7.5 g of sodium carboxymethyl cellulose and slowly and evenly sprinkle it into the beaker while stirring at 300 rpm (to ensure that the sodium carboxymethyl cellulose is evenly dispersed in the water). After the addition is complete, adjust the stirring speed to about 500 rpm and stir for 12.5 h. ② Add 200 mL of purified water to a 250 mL beaker and heat it to 70 °C in a water bath. Weigh out 1.5 g of sodium hyaluronate and slowly and evenly sprinkle it into the beaker while stirring (to ensure that the sodium hyaluronate is evenly dispersed in the water). Continue stirring for 20 min while keeping the temperature warm. After there are no obvious particles in the solution, transfer it to an automatic rotary stirrer (speed: about 500 rpm) and continue stirring for 1.5 h. ③ After mixing the materials in ① and ② evenly, sterilize them in a 100 °C autoclave for 10 min to obtain the ointment matrix.

[0077] (2) Preparation of peptide solution: Under sterile conditions, add 200mL of purified water to a 250mL beaker, weigh 90g of pineapple peptide, add it and stir to dissolve, thus obtaining peptide solution.

[0078] (3) Pretreatment of other excipients: Under sterile conditions, take 30g of arbutin and dissolve it in 40mL of purified water at about 45℃. Then, take another 9g of azone and dissolve it in 15mL of ethanol. Mix the two evenly and let them stand for later use.

[0079] (4) Preparation of ointment: Under sterile conditions, the peptide solution from step (2), the excipient solution from step (3), and 30g of vitamin E were added to the prepared matrix in sequence, and the weight was adjusted to 1000g with purified water. After stirring for 45min to ensure complete mixing of the materials, the mixture was placed in a high-pressure steam sterilizer and sterilized at 80℃ for 15min. After cooling, the mixture was dispensed into PE bottles sterilized by ultraviolet light, 10g / bottle, for a total of 94 tubes of pineapple peptide scar ointment.

[0080] Comparative Example 1

[0081] Preparation of negative control samples (without bromelain)

[0082] (1) Preparation of the matrix: ① Add 150 mL of purified water to a 250 mL beaker and place it under an automatic rotary stirrer. Weigh out 7.5 g of sodium carboxymethyl cellulose and slowly and evenly sprinkle it into the beaker while stirring at 300 rpm (to ensure that the sodium carboxymethyl cellulose is evenly dispersed in the water). After the addition is complete, adjust the stirring speed to about 500 rpm and stir for 12.5 h. ② Add 200 mL of purified water to a 250 mL beaker and heat it to 70 °C in a water bath. Weigh out 1.5 g of sodium hyaluronate and slowly and evenly sprinkle it into the beaker while stirring (to ensure that the sodium hyaluronate is evenly dispersed in the water). Continue stirring for 20 min while keeping the temperature warm. After there are no obvious particles in the solution, transfer it to an automatic rotary stirrer (speed: about 500 rpm) and continue stirring for 1.5 h. ③ After mixing the materials in ① and ② evenly, sterilize them in a 100 °C autoclave for 10 min to obtain the ointment matrix.

[0083] (2) Preparation of peptide solution: Under sterile conditions, add 200mL of purified water to a 250mL beaker, weigh out 30g of thymosin peptide, 50g of sheep placental extract and 40g of bromelain, add them in sequence and stir to dissolve, and obtain a peptide mixed solution.

[0084] (3) Pretreatment of other excipients: Under sterile conditions, take 30g of arbutin and dissolve it in 40mL of purified water at about 45℃. Then, take another 9g of azone and dissolve it in 15mL of ethanol. Mix the two evenly and let them stand for later use.

[0085] (4) Preparation of ointment: Under sterile conditions, the peptide solution from step (2), the excipient solution from step (3), and 30g of vitamin E were added to the prepared matrix in sequence, and the weight was adjusted to 1000g with purified water. After stirring for 45min to ensure complete mixing of the materials, the mixture was placed in a high-pressure steam sterilizer and sterilized at 80℃ for 15min. After cooling, the mixture was dispensed into PE bottles sterilized by ultraviolet light, 10g / bottle, for a total of 92 tubes of pineapple peptide scar ointment.

[0086] The product formulations of Examples 1-4 and Comparative Example 1 are summarized in Table 1.

[0087] Table 1. Formulation information of various samples of pineapple peptide ointment

[0088]

[0089] Example 5

[0090] Pineapple Peptide Ointment Quality Testing

[0091] The results of the physicochemical index detection and analysis of Examples 1 to 4 above are shown in Table 2.

[0092] Table 2. Quality test results of various samples of pineapple peptide ointment

[0093]

[0094] The comparative analysis in the table above shows that the pineapple peptide ointment prepared in this invention has good quality parallelism and the process is stable.

[0095] Example 6

[0096] Experimental study on the efficacy of pineapple peptide ointment in promoting scar repair and inhibiting scar hyperplasia.

[0097] (1) Preparation of a mouse scald scar model: 150 healthy SPF-grade Kunming mice (half male, half female, 18-22g each) were selected. 24 hours before the experiment, the backs of the mice were shaved, and sodium sulfide was used to remove hair until no hair remained. During the experiment, the mice were anesthetized with sodium pentobarbital. The limbs and tails of the mice were fixed to a clean bench. After cleaning the shaved areas with physiological saline, a bottomless plastic cylinder (1.2cm in diameter and 4cm in height) was placed in close contact with the skin at the shaved area (the contact pressure ensured no water leakage and no mechanical damage to the skin). Boiling water (2mL) at 100℃ was rapidly injected into the plastic tube and held for 10 seconds. After the injury, the mice were housed separately, with dry and clean bedding, good ventilation, and sufficient feed and water. No animals died during the experiment.

[0098] (2) Experimental Methods: 140 mice with scalded wounds that had healed for one week were randomly divided into a treatment group (n=100), a positive control group (n=20), and a blank control group (n=20). Before each application of the medication, the scar area was cleaned with physiological saline. Samples of pineapple peptide ointment (Examples 1-4), a sample without pineapple peptide (Comparative Example 1), and a positive control sample (silicone preparation) were evenly applied to the healed skin surface of the mice. The ointment was gently massaged with fingertips for 5-10 minutes to ensure full absorption. The treatment was repeated twice daily, morning and evening, for 90 days. The blank control group had their scars cleaned with physiological saline concurrently.

[0099] (3) Observation indicators and judgment criteria

[0100] Observation indicators: pigmentation, softness, thickness of scar sites in mice of each group, and serum concentration of anti-vascular endothelial growth factor.

[0101] Judgment criteria: The Vancouver Scar Rating Scale (VSS, see Table 3) was used to assess the scar repair status in mice. Scores ranging from 0 to 15 were assigned based on the scoring criteria in the scale, with higher scores directly proportional to scar severity. Hypertrophic scars are a manifestation of excessive tissue repair after burns, trauma, or lesions. Early hypertrophic scars are characterized by vigorous vascular proliferation. Studies have shown a positive correlation between serum anti-vascular endothelial growth factor (VEGF) concentration and scar hyperplasia. This experiment measured the serum VEGF concentration in mice to determine the degree of scar hyperplasia at the wound healing site. The results are shown in Table 4.

[0102] Table 3. Vancouver Scar Rating Scale (Partial)

[0103]

[0104]

[0105] Table 4. Comparison of examination indicators before and after the experiment in each group of mice

[0106]

[0107]

[0108] Note: a. Compared with the blank control group: P # <0.05, P ## <0.01; b. Within-group comparison before and after the experiment: P * <0.05, P ** <0.01.

[0109] Table 4 shows that before the experiment, compared with the blank control group, there were no significant differences in VSS scores, scar thickness, and serum VEGF concentration among the mice in each group, indicating that the baseline interference factors of the experimental mice were small. Within each group, before and after the experiment, the blank control group showed a slight decrease in VSS scores and scar thickness (P>0.05) and a significant increase in serum VEGF concentration (P<0.05), while the positive control group and experimental groups 1-5 showed significant decreases in VSS scores, scar thickness, and serum VEGF concentration (P<0.05, P<0.01). After the experiment, compared with the blank control group, the positive control group and experimental groups 1-4 showed significant decreases in VSS scores, scar thickness, and serum VEGF concentration (P<0.05, P<0.01), while experimental group 5 (without pineapple peptide sample) showed a slight decrease, but it was not significant. The experimental results above show that, without external intervention, mice exhibit some scar repair, but this is not significant. The pineapple peptide compound product, however, has a more significant effect on scar repair in mice, and its efficacy is superior to silicone-based preparations. Furthermore, data indicates that fibroblasts in scars interact and regulate with vascular endothelial cells through VEGF, promoting abnormal blood vessel growth and scar hyperplasia in pathological scars. In the experiment, without external intervention, the VEGF concentration in mice remained consistently high, indicating scar hyperplasia. Pineapple peptide and its compound substances significantly reduced serum VEGF concentration, thereby inhibiting scar hyperplasia in mice. In conclusion, the pineapple peptide ointment in this experiment promotes scar repair and inhibits scar hyperplasia in mice.

[0110] Meanwhile, a comprehensive comparative analysis of the experimental results of experimental groups 1, 2, 3, and 5, and experimental groups 2, 4, and 5 showed that pineapple peptide plays a dominant role in the efficacy of pineapple peptide ointment in promoting scar repair and inhibiting scar hyperplasia in mice.

[0111] It is evident that the pineapple peptide ointment of the present invention has the effects of reducing pigmentation, smoothing scar thickness, softening scar texture, and inhibiting scar hyperplasia.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A pineapple peptide ointment, characterized in that, The raw materials of the pineapple peptide ointment include pineapple peptide, thymosin peptide, sheep placenta extract, bromelain, vitamin E, arbutin, moisturizer, thickener, penetrant, and purified water; wherein, by weight fraction, the proportions are: 9-10% pineapple peptide, 3-4% thymosin peptide, 5-6% sheep placenta extract, 4-5% bromelain, 3-4% vitamin E, 3-4% arbutin, 0.15-0.2% moisturizer, 0.75-1.0% thickener, and 0.9-1.0% penetrant. The remainder is purified water; The moisturizer is selected from one of sodium hyaluronate, glycerol, butylene glycol, propylene glycol, sorbitol, xylitol, polyethylene glycol, polyglycerol, sodium lactate, pyrrolidone carboxylic acid, or urea; The penetrant is selected from one of azone, isosorbide dimethyl ether, thiazoline, inositol, or tetrahydropenetrose; The preparation method of the pineapple peptide is as follows: 1) Take fresh pineapple fruit and stem, wash and drain them, then crush them with a grinder, then homogenize them in a homogenizer at 2-8℃ and treat them with ultrasound to obtain a homogenate. After removing the fiber residue by coarse filtration with a filter cloth, take the filtrate. 2) Adjust the pH of the filtrate to 4.5-5.5 with sodium hydroxide solution, heat it in a water bath to 45-55℃, add 0.1%-0.2% of cellulase by weight of the slurry, keep it warm for 1-2 hours, and after the reaction is complete, heat it in a water bath to 75-85℃ and keep it warm for 10-20 minutes. 3) After cooling to 50–60°C from room temperature, adjust the pH to 6.0–7.0 with sodium hydroxide solution, and add 0.2%–0.3% by weight of the slurry. Compound flavor protease, incubate for 2-4 hours, after the reaction is complete, heat in a water bath to 75-80℃ and incubate for 10-15 minutes; 4) The enzymatic hydrolysate is concentrated by ultrafiltration using a series of 20K / 10K / 5KDa ultrafiltration membranes; 5) The concentrated solution was placed in a sample tray, pre-cooled in a refrigerator for 30 minutes, and then placed in a vacuum freeze dryer. It was freeze-dried according to the preset freeze-drying curve to obtain pineapple peptide. The method for preparing the thymocyte protein peptide is as follows: a) Take fresh, healthy calf thymus and wash it several times with 0.9% physiological saline; cut it into small pieces with a grinder, and then homogenize it in a homogenizer at 2-8℃ to obtain a homogenate. b) After freezing the homogenate in a freezer at -18°C, take it out and heat it in a water bath at 40°C to dissolve it. After it is completely dissolved, put it back into the freezer at -18°C and freeze it. Repeat this freeze-thaw cycle at least 3 times. c) Adjust the pH of the above freeze-thawed solution to 2.0-3.0 with hydrochloric acid solution, heat it in a water bath to 35-45°C, add 0.4%-0.6% pepsin by weight of the slurry, and keep it warm for 2-4 hours; d) After the reaction is complete, heat the mixture in a water bath to 75-85°C and keep it at that temperature for 10-20 minutes. Then, concentrate the mixture by ultrafiltration using a series of 20K / 10K / 5KDa ultrafiltration membranes. e) The concentrate is placed in a sample tray, pre-cooled in a refrigerator for 30 minutes, and then placed in a vacuum freeze dryer. The solution is freeze-dried according to the preset freeze-drying curve to obtain thymocyte peptides.

2. The pineapple peptide ointment according to claim 1, characterized in that, The thickener is selected from one of sodium carboxymethyl cellulose, propylene glycol alginate, polyoxyethylene, sodium polyacrylate, casein, or polyvinylpyrrolidone.

3. The pineapple peptide ointment according to claim 1, characterized in that, The cellulase used in step 2) has an enzyme activity of 100,000 u / g; the complex flavor protease used in step 3) has an enzyme activity of 50,000 u / g.

4. The pineapple peptide ointment according to claim 1, characterized in that, The pepsin used in step c) has an enzyme activity of 12,000 u / mg.

5. A method for preparing pineapple peptide ointment as described in any one of claims 1-4, characterized in that, The preparation method includes the following steps: S1. Preparation of the base: According to the formula, the thickener and humectant are dissolved in water to form a gel-like state. After being mixed evenly, the mixture is sterilized in a steam sterilizer to serve as the ointment base. S2. Under sterile conditions, pineapple peptide, thymosin peptide, sheep placental extract and bromelain were dissolved in water in sequence to prepare a peptide solution. S3. Pretreatment of other excipients: Under sterile conditions, arbutin and the penetrant are dissolved separately in purified water and ethanol, and then mixed to obtain an excipient solution; S4. Preparation of ointment: Under sterile conditions, the peptide solution obtained in s2, the excipient solution obtained in s3, and vitamin E are added sequentially to the ointment base obtained in s1. The contents are weighed and calibrated, stirred and mixed, and sterilized in a steam sterilizer for 10-20 minutes. After cooling, the ointment is filled to obtain pineapple peptide ointment.

6. The method for preparing pineapple peptide ointment according to claim 5, characterized in that, In S1, the steam sterilization temperature is 100℃; in S4, the steam sterilization temperature is 80-85℃.

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