Novel polypeptides for promoting tissue repair and their use

Abstract

Novel polypeptides and their uses for promoting tissue repair are disclosed. [Solution] The novel polypeptide can promote the proliferation of human immortalized epidermal cells and the migration of human epidermal fibroblasts at low concentrations, promote the repair of wounds and ulcers, and improve the aesthetic appearance of the skin. It has low toxicity and side effects and good prospects for use, and can be used in the preparation of medicines and medical devices for the treatment of wounds, burns and ulcers, or daily chemical products for improving the aesthetic appearance of the skin.

Description

Technical Field

[0001] The present invention relates to the fields of pharmaceuticals, medical devices, and daily chemical products, and particularly relates to novel polypeptides for promoting tissue repair and their uses.

Background Art

[0002] With the development of society and the acceleration of aging, tissue trauma includes not only external injuries such as burns and wounds, but also intractable wounds and ulcers such as gastric ulcers, oral ulcers, diabetic ulcers, autoimmune skin ulcers, venous stasis ulcers, and pressure ulcers due to long-term bedridden. The pathological conditions of intractable wounds and ulcers are complex, the course is long, the treatment is difficult, and the treatment cost is high, which brings physical, mental burdens and economic burdens to patients. At present, growth factor preparations are widely used in the treatment of intractable wounds and ulcers and show good therapeutic effects. Among them, epidermal growth factor, basic fibroblast growth factor, vascular endothelial growth factor, etc. are all important for the healing of wounds and ulcers. However, these endogenous factors have high preparation costs and poor stability, so their clinical applications are limited. Therefore, in order to treat tissue trauma, intractable wounds and ulcers, it is highly demanded to find and develop active substances with high activity, low manufacturing cost, and excellent stability.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The object of the present invention is to overcome the above-mentioned drawbacks of the prior art and provide a novel polypeptide that can promote tissue repair and its uses.

Means for Solving the Problems

[0004] To achieve the above object, the present invention uses the following technical means.

[0005] The novel polypeptides for promoting tissue repair, named TRF1 to TRF12, have a structure represented by Formula I: JPEG0007855811000001.jpg In formula I of 1963, R1 = H or Ala or Gly or Ala-Ala- or Val-Ala-; R2 = Asn or Asp; R3=Ser or Tyr or -Gly-Ser-Tyr or -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr or -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr- His-Val-Arg or -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His-Val-Arg-Glu-Tyr-Pro-Ala-Gly-Val-Ser-Ala-Ala.

[0006] Furthermore, in preferred embodiments of the present invention, the novel polypeptide compounds include, but are not limited to, the following TRF1 to TRF12. JPEG0007855811000002.jpg91154

[0007] The novel polypeptides (TRF1 to TRF12) can be prepared using conventional methods, chemically synthesized using a polypeptide synthesizer, or biosynthesized by predicting the nucleotide sequence from the polypeptide sequence and cloning it into an expression vector.

[0008] Another object of the present invention is to disclose the use of the novel polypeptides (TRF1 to TRF12) in the preparation of products for promoting tissue repair or improving skin aesthetics.

[0009] Preferably, the novel polypeptides (TRF1 to TRF12) are used in the preparation of products for the treatment of wounds, burns, ulcers, or for improving the appearance of skin.

[0010] Preferably, the product is a pharmaceutical, a medical device, or a daily chemical product.

[0011] Preferably, the pharmaceutical product, medical device, or daily chemical product contains an effective amount of one or more novel polypeptides (TRF1 to TRF12), or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, with the remainder being an additive or other compoundable agent.

[0012] The aforementioned additives are conventional additives such as solvents, disintegrants, flavor modifiers, preservatives, colorants, and binders.

[0013] The aforementioned compoundable agents include other natural pharmaceuticals, chemicals, and biological preparations.

[0014] The aforementioned pharmaceuticals, medical devices, or daily chemicals may be in the form of tablets, capsules, injections, liposome nanoparticles, release control agents, gel creams, ointments, liniments, patches, creams, cleansers, lotions, gels, toners, and the like. [Effects of the Invention]

[0015] The present invention has the following advantages and effects compared to the prior art. (1) The polypeptides (TRF1 to TRF12) according to the present invention are polypeptides with novel structures, all of which contain disulfide bonds, are structurally stable and resistant to degradation. (2) The novel polypeptides (TRF1 to TRF12) according to the present invention significantly promote the proliferation of human immortalized epidermal cells (HaCAT) and significantly promote the migration of human dermal fibroblasts (HSF), and have a tissue repair promoting effect. (3) The novel polypeptides (TRF1 to TRF12) according to the present invention can be used to promote tissue repair and treat peptic ulcers and oral ulcers, and have the effect of removing wrinkles, scars and pigmentation. (4) The novel polypeptides (TRF1 to TRF12) according to the present invention have low toxicity, and no obvious toxic side effects were observed with oral administration of 500 mg / kg. (5) The novel polypeptides (TRF1 to TRF12) according to the present invention can be chemically synthesized or biosynthesized and are easy to prepare in large quantities. [Brief explanation of the drawing]

[0016] [Figure 1] Mass spectra of the novel polypeptide TRF1. A is a diagram showing its ESI-MS, B is a diagram showing its MS2 (m / z 50 - 580), and C is a diagram showing its MS2 (m / z 580 - 1110). [Figure 2] Mass spectra of the novel polypeptide TRF2. A is a diagram showing its ESI-MS, B is a diagram showing its MS()2 (m / z 50 - 420), C is a diagram showing its MS2 (m / z 390 - 810), and D is a diagram showing its MS2 (m / z 810 - 1170). [Figure 3] Mass spectra of the novel polypeptide TRF3. A is a diagram showing its ESI-MS, B is a diagram showing its MS2 (m / z 50 - 600), and C is a diagram showing its MS2 (m / z 600 - 1300). [Figure 4] Mass spectra of the novel polypeptide TRF4. A is a diagram showing its ESI-MS, B is a diagram showing its MS2 (m / z 50 - 510), and C is a diagram showing its MS2 (m / z 510 - 1250). [Figure 5] Mass spectra of the novel polypeptide TRF5. A is a diagram showing its ESI-MS, B is a diagram showing its MS2 (m / z 50 - 650), and C is a diagram showing its MS2 (m / z 620 - 1200). [Figure 6] Mass spectra of the novel polypeptide TRF6. A is a diagram showing its ESI-MS, B is a diagram showing its MS2 (m / z 50 - 400), C is a diagram showing its MS2 (m / z 400 - 900), and D is a diagram showing its MS2 (m / z 850 - 1800). [Figure 7] Mass spectra of the novel polypeptide TRF7. A is a diagram showing its ESI-MS, B is a diagram showing its MS2 (m / z 50 - 400), C is a diagram showing its MS2 (m / z 400 - 970), and D is a diagram showing its MS2 (m / z 970 - 1700). [Figure 8] Mass spectra of the novel polypeptide TRF8. A is a diagram showing its ESI-MS, B is a diagram showing its MS2 (m / z 50 - 620), and C is a diagram showing its MS2 (m / z 620 - 1150). [Figure 9] Mass spectrum of the novel polypeptide TRF9. A shows its ESI-MS, B shows its MS2 (m / z 50 - 420), C shows MS2 (m / z 390 - 810), and D shows MS2 (m / z 800 - 1200). [Figure 10] Mass spectrum of the novel polypeptide TRF10. A shows its ESI-MS, B shows its MS2 (m / z 50 - 500), and C shows MS2 (m / z 450 - 1250). [Figure 11] Mass spectrum of the novel polypeptide TRF11. A shows its ESI-MS, B shows its MS2 (m / z 50 - 420), C shows MS2 (m / z 390 - 820), and D shows MS2 (m / z 800 - 1270). [Figure 12] Mass spectrum of the novel polypeptide TRF12. A shows its ESI-MS, B shows its MS2 (m / z 50 - 380), C shows MS2 (m / z 380 - 920), and D shows MS2 (m / z 920 - 1560). [Figure 13] Figure showing the results of analyzing the effect of the novel polypeptides TRF1 - TRF12 on the proliferation of HaCAT cells. [Figure 14] Figure showing the results of analyzing the effect of the novel polypeptides TRF1 - TRF12 on the migration of HSF cells.

Mode for Carrying Out the Invention

[0017] Hereinafter, the present invention will be described in detail with reference to the drawings according to embodiments, but the embodiments of the present invention are not limited thereto.

[0018] Example 1 Synthesis of the novel polypeptides TRF1 - TRF12 (1) Solid-phase synthesis of the novel polypeptide TRF1 A tyrosine-Wang resin containing an Fmoc group and a side-chain protecting group at a concentration of 0.3-0.5 mmol / g was placed in a solid-phase synthesizer, and 20 mL of N,N-dimethylformamide was added to completely dissolve it. After extracting the solvent, 20% piperidine (dissolved in N,N-dimethylformamide by volume) was added, and the mixture was stirred for about 10 minutes. The solvent was then extracted, and this procedure was repeated twice to completely remove the Fmoc group. After that, 20 mL of N,N-dimethylformamide was added for washing, the solvent was extracted, and this procedure was repeated three times. 0.5 mmol of cysteine ​​containing an Fmoc group and a side-chain protecting group was added, followed by equal volumes of HBTU / N,N-dimethylformamide and N,N-diisopropylethylamine / N,N-dimethylformamide solution. The mixture was reacted under an N2 atmosphere for 40-60 minutes. Unreacted drugs and reagents were eluted with N,N-dimethylformamide and detected with ninhydrin. Subsequently, the remaining amino acids were bonded using a similar method, and peptides were synthesized bound to the resin through steps such as deprotection, activation, condensation, and washing. The peptides on the resin were treated with Clevage solution (trifluoroacetic acid / triisopropylsilane / pure water / 1,2-ethanedithiol, in a volume ratio of 94:2.5:2.5:1) for 2-3 hours, followed by ice-cold ethyl ether and centrifugation to obtain the precipitate at the bottom. The precipitate was removed, the remaining trifluoroacetic acid was removed, and a predetermined amount of iodine solution was added. The mixture was stirred at room temperature for about 1 hour to react and obtain the crude product of the target polypeptide. The crude polypeptide product was dissolved in a 20% aqueous acetonitrile solution, filtered, and purified by high-performance liquid chromatography at detection wavelengths of 214 nm, 254 nm, and 280 nm, with acetonitrile-water mobile phase (v / v: 10:90~50:50) and a flow rate of 1 mL / min. After collecting the desired peaks, the product was concentrated and freeze-dried to obtain the purified polypeptide. (2) Solid-phase synthesis of novel polypeptides TRF2 to TRF12 The synthesis process for polypeptides TRF2 to TRF12 was the same as that for TRF1. 0.3 to 0.5 mmol / g of amino acids (the first one at the carboxyl terminus) having an Fmoc group and a side-chain protecting group, along with Wang resin, were placed in a solid-phase synthesizer. Through deprotection, activation, condensation, and washing steps, the amino acids in the sequence were sequentially linked to obtain polypeptides bound to the resin. These polypeptides were then cleaved and purified to obtain purified polypeptides.

[0019] Example 2: Mass spectrometry identification of novel polypeptides TRF1-TRF12 TRF1 to TRF12 polypeptides were weighed and dissolved in deionized water to prepare 0.1 mg / mL sample solutions. Each sample solution was injected into an AB Sciex TripleTOF® 5600+ mass spectrometer for analysis. Mass range (m / z): 50-2000 Da, uptake mode: positive ion mode, scan mode: selected ion scan, capillary voltage: 3.0 kV, ion source temperature: 110°C, collision energy range: 50-80 V. As shown in Figures 1-12, a secondary scan was performed on the precursor ions, and the amino acid residue sequences of the novel polypeptides were identified by analyzing the b and y ions based on the fragmentation of the secondary mass spectrum.

[0020] Example 3: Promotion of HaCAT cell proliferation by novel polypeptides TRF1-TRF12 (1) Cell inoculation: Cells in the logarithmic growth phase were selected, digested, and counted. The required number of cells were collected and an appropriate amount of culture medium was added to form 1 × 10⁻⁶ cells. 4 The solution was diluted to 1 / mL. The cells were homogenized using a pipette gun, added to a 96-well plate (100 μL / well), and cultured in a CO2 incubator. (2) Addition of drugs: After the cells adhered to the wall, the old medium was removed, and blank medium was added to the control group, EGF to the positive control group, and TRF1 to TRF12 at different concentrations to the drug groups. PBS was added around the plate, and 6 wells were used per group. The cells were incubated in an incubator for 48 hours. (3) Addition of MTT: The old culture medium was aspirated and removed, and 30 μL of prepared MTT (light-shielded) was added to each well. The wells were then placed in an incubator and incubated for 4 hours to form formazan crystals. (4) Measurement of OD value: After removing the MTT, 100 μL of DMSO solution was added to each well, and the formazan was thoroughly dissolved. The absorbance was then detected using a multi-functional microplate reader (detection wavelength: 595 nm). Cell viability was calculated, and the test was repeated at least three times. As shown in Figure 13, the novel polypeptides TRF1 to TRF12 showed a significant proliferation-promoting effect on HaCAT cells at low concentrations (0.08 to 2.00 μg / mL) (cell proliferation rate of 114 ± 5% to 135 ± 3%).

[0021] Example 4: Promotion of HSF cell migration by novel polypeptides TRF1-TRF12 (1) Cell inoculation and drug addition: Transwell was UV sterilized in a clean bench for 30 minutes and then placed in a 24-well plate. After the cells were digested, they were counted and added to the culture medium in a 2 × 10⁶ 5 The cells were diluted to a density of cells / mL. The test drugs TRF1-TRF12 and the positive control drug were added, and the mixture was diluted with drug-containing cell saturates of different concentrations. 100 μL of drug-containing cell saturate was added to the upper chamber of the drug-administered group, and 100 μL of cell saturate was added to the blank group. Then, 600 μL of blank medium was added to each chamber, and the cells were cultured in an incubator. (2) Fixation, staining, and photography: After culturing, the culture medium was discarded, washed with PBS, and the cells in the lower chamber were fixed with 4% paraformaldehyde (for about 30 minutes). After fixation, the cells were washed twice with PBS, and 600 μL of staining solution was added to the lower chamber of each well and stained for 20 minutes. The crystal violet was collected and washed with PBS. Cells that did not migrate well were carefully wiped off by pressing a cotton swab against the membrane of the upper chamber, and after air drying, 5 random areas were photographed for each chamber using an inverted fluorescence microscope. The average number of cells that migrated last was statistically analyzed using GraphPad Prism 6.0. The test results showed that the novel polypeptides TRF1-TRF12 (2 μg / mL) significantly promoted HSF cell migration. After 48 hours of action, the average number of migrating cells in the drug group ranged from 578 ± 9 to 396 ± 14, which was significantly higher than the control group's 132 ± 10, demonstrating statistical significance (P<0.05). The results are shown in Figure 14.

[0022] Example 5: Repair effect of novel polypeptide TRF7 on mouse skin wounds Fifteen male Kunming mice aged 8-10 weeks (purchased from the Guangdong Provincial Medical Experimental Animal Center) were selected and housed in separate cages. They were randomly divided into a control group, a positive drug administration group (Rehabilitation Solution), a low-dose TRF7 administration group (0.5 mg / mL), and a high-dose TRF7 administration group (2.0 mg / mL). The mice were anesthetized, their backs were depilated and disinfected, and a 6 mm diameter punch was used to make holes in the exposed skin on the backs of the mice. A circular area of ​​skin, including the epidermis and dermis, was excised, and a transparent oxygen-permeable dressing was applied to the wound site to provide shielding and protection. In the control group, saline solution was administered, the transparent oxygen-permeable dressing was gently lifted, 0.1 mL of liquid was added to the wound, and a new dressing was applied. The drugs were administered twice a day for 10 consecutive days, and the change in wound size was measured daily. The mice were executed 10 days after administration. The wound healing rate is calculated as: Wound healing rate % = (S1 - S n ) / S1(S1 is the wound area on the first day, S n The wound area on day n was calculated using the formula (where n is the wound area). The results are shown in Table 1. JPEG0007855811000003.jpg60164 Note: Significant difference observed compared to the model group (*P<0.05) The test results showed that the novel polypeptide TRF7 significantly promoted the healing of skin wounds in mice, with a significant difference compared to the control group and superior to the rejuvenation solution.

[0023] Example 6: Repair effect of novel polypeptide TRF7 on mouse skin burns Fifteen male Kunming mice aged 8-10 weeks (purchased from the Guangdong Provincial Center for Experimental Animal Health) were selected, housed in separate cages, and allowed to feed freely. Food intake was stopped the day before the experiment. Hair removal was performed by applying a 13% Na2S solution to the mice. A 20g weight was placed in a water bath, heated until boiling, and maintained for 10 minutes. Then, the upper end of the weight was grasped with pliers and quickly placed on the mouse's skin for 5 seconds, applying slight pressure to create a 2cm x 2cm superficial second-degree burn model. The following day, 0.1mL each of TRF7 (0.5mg / mL or 2.0mg / mL) with different solubility, physiological saline, and a rejuvenating solution were dropped onto the burn, and the medication was changed once daily. Burn healing was observed on days 3, 5, and 10. The burn healing rate was calculated according to the method of Example 5. The results are shown in Table 2. JPEG0007855811000004.jpg61165 Note: Significant difference observed compared to the model group (*P<0.05) The test results showed that the novel polypeptide TRF7 significantly promoted the healing of skin burns in mice, with a significant difference compared to the control group and superior to the conventional healing solution.

[0024] Example 7: Protective effect of novel polypeptide TRF7 against stress-induced gastric ulcers in rats Method: Twenty male SD rats weighing 180-210g (purchased from the Guangdong Provincial Medical Experimental Animal Center) were collected and randomly divided into a blank group, a model group, a sucralfate administration group (1g / kg, positive drug administration group), a TRF7 low-dose administration group (12.5mg / kg), and a TRF7 high-dose administration group (50.0mg / kg). The drug administration groups received forced oral administration for 7 consecutive days. Before modeling, the rats in each group were fasted for 24 hours without deprivation of water. Except for the control group, the rats were modeled using the restraint water immersion method. The rats in each group were fixed to a rat board and immersed vertically with their heads up in a constant temperature (20°C) water bath for 8 hours, with the water level at the same height as the rat's xiphoid process. At the end of stress modeling, the rats were dislocated, dissected, and the pylorus was ligated. The stomach was perfused with 2 mL of 10% formaldehyde solution, the cardia was ligated, and the stomach body was removed and fixed in formaldehyde solution for 15 minutes. The stomach was cut along the greater curvature, rinsed with saline solution, unfolded, and the damage to the gastric mucosa was observed, and the gastric ulcer index was calculated. The ulcer index (UI) was calculated according to Guth's criteria, with 1 point for petechiae, 2 points for linear hemorrhages less than 1 mm in length, 3 points for 1-2 mm, 4 points for 2-4 mm, 5 points for greater than 4 mm, and ×2 points for width greater than 1 mm. The results are shown in Table 3. JPEG0007855811000005.jpg44142 Note: Significant difference observed compared to the model group (*P<0.01) The test results showed that the novel polypeptide TRF7 demonstrated a significant protective effect against stress-induced gastric ulcers in rats, with a statistically significant difference compared to the model group, and this protective effect was superior to that of the positive drug sucralfate.

[0025] Example 8: Protective effect of novel polypeptide TRF7 on oral ulcers in rats Test Method: Fifteen healthy SD rats weighing 180-210g (purchased from the Guangdong Provincial Medical Experimental Animal Center) were randomly divided into a model group, a positive drug administration group (Kangfu Xin Liquid), a TRF7 low-dose administration group (0.5 mg / mL), and a high-dose administration group (2.0 mg / mL), and were modeled by chemical cauterization. A 5 mm diameter plastic tube was taken, a small cotton ball soaked in 95% phenol solution was placed on one end of the tube, and the other end of the cotton ball was fixed to the left buccal mucosa of anesthetized rats. After holding for 60 seconds, the cauterized surface was washed with physiological saline. After 24 hours, the formation of a round ulcer was observed on the left side of the test site, with a yellowish-white pustule on its surface. The surrounding area was clearly demarcated, the mucosa was red, swollen, and congested, and the ulcer surface diameter was >3 mm. The model was successfully established. In the test group, model group, and positive drug group, 0.1 mL each of the novel polypeptide solution, physiological saline, and rejuvenating solution were administered twice daily to the ulcerated surface. Ulcer healing was observed after 1 day, 3 days, and 5 days. The scoring criteria for ulcers were as follows: 1 point for no congestion or edema and an ulcer surface diameter of less than 1 mm; 2 points for mild congestion and edema and an ulcer surface diameter of 1-2 mm; 3 points for moderate congestion and edema and an ulcer surface diameter of 2-3 mm; and 4 points for severe congestion and edema and an ulcer surface diameter of 3 mm or more. The results are shown in Table 4. JPEG0007855811000006.jpg66164 Note: Significant difference observed compared to the model group (*P<0.05) The test results showed that the novel polypeptide TRF7 significantly promoted the healing of oral ulcers in rats, with a significant difference compared to the model group, and was superior to the rejuvenation liquid.

[0026] Example 9: Acute toxicity test of novel polypeptides TRF1-TRF12 Test Method: Kunming mice weighing 18-22g (purchased from the Guangdong Provincial Medical Experimental Animal Center) were collected and randomly divided into groups of 10. Different doses of TRF1-TRF12 were administered orally, and the median lethal dose (LD) was determined based on the survival rate of the mice. 50 The median lethal dose was calculated using the following formula: Median Lethal Dose (mg / kg) = (Dose at which half of the mice die / Body weight of the affected mice). Test results: No deaths or serious side effects were observed in mice at a dose of 500 mg / kg for any of the novel polypeptides TRF1-TRF12. 50 It was shown to have low toxicity at doses exceeding 500 mg / kg (Table 5). JPEG0007855811000007.jpg34161

[0027] Example 10: Skin safety evaluation of novel polypeptide TRF7 Participants: 20 individuals aged 18-55, 10 women and 10 men. All participants had healthy skin, no history of skin allergies, and met the criteria for voluntary enrollment. Test Method: A suitable patch device was selected, and using a closed patch test method, approximately 0.020-0.025 mL (2 mg / mL) of polypeptide TRF7 was dropped onto the patch device, and a special topical tape was applied to the subject's back. After 24 hours, the test sample was removed, and skin reactions were observed at 0.5, 6, 12, 24, and 48 hours after removal. The results were recorded according to the skin reaction grading criteria of the "Cosmetic Hygiene Standards". Test Results: In this study, skin reactions were observed in 20 subjects via patch tests at 0.5, 6, 12, 24, and 48 hours. No adverse skin reactions were observed, demonstrating that the novel polypeptide TRF7 of the present invention is safe for the skin.

[0028] Example 11: Preparation of Tablets 0.1g of polypeptide TRF7, 40g of lactose, 60g of starch slurry, and 0.2g of magnesium stearate were taken, mixed, sieved, dried, and formed into tablets. Each tablet contains 0.001g of TRF7.

[0029] Example 12 Preparation of injectable drug 0.1 g of polypeptide TRF7 and 50 g of propylene glycol were ground, diluted with 100 mL of sterile water for injection, and thoroughly mixed. Then, 9 g of sodium chloride was added and dissolved, and then sterile water for injection was added to make a total of 1000 mL. The pH was adjusted to 5.5-6.5, filtered, potted, and sterilized to obtain 1000 vials of the injectable solution.

[0030] Example 13 Preparation of Solid Lipid Nanoparticles 0.1 g of polypeptide TRF7 was taken and dissolved in 25 mL of ethanol with 500 mg of soy lecithin. 200 mg of stearic acid and 500 mg of soy lecithin were dissolved in 25 mL of cyclohexane and thoroughly mixed and stirred. The mixture was evaporated under reduced pressure in a 37°C constant temperature water bath to remove the organic solvent, allowing the drug and additives to form a uniform lipid film on the flask wall. The solution was then placed in a vacuum desiccator overnight to completely remove the organic solvent. 3750 mg of polyethylene glycol monostearate was dissolved in 175 mL of water with stirring, added to the film, and sonicated for 10 minutes. The volume was then adjusted to 250 mL to obtain a pale yellow, transparent solution. This solution was freeze-dried to obtain a freeze-dried powder. The powder was ground in a ball mill for 24 hours to obtain nanoparticles of uniform size, which were mixed and packaged in small portions. Each bag contains 0.001 g of TRF7.

[0031] Example 14 Preparation of controlled-release capsules 0.1 g of polypeptide TRF7 was sequentially mixed with 40 g of lactose and 10 g of starch slurry, and the mixture was directly loaded into a rotary granulation and coating machine to prepare pellets. A suspension of plasticized ethylcellulose coating agent, diluted to 15% by mass, was sprayed onto the rotating bed of polypeptide pellets. During spraying, the pellets were film-coated with a dispersion carrier made of poloxamer 188, forming sustained-release pellets with an average particle size of approximately 450 μm. The mixture was thoroughly mixed and filled into capsules, with each capsule containing 0.001 g of TRF7.

[0032] Example 15 Preparation of topical gel cream 2 g of glycerin was weighed, and 0.75 g of polyacrylic acid and 0.1 g of aluminum hydroxide were added sequentially and mixed thoroughly. Then 0.2 g of polypeptide TRF7 was added and mixed thoroughly under vacuum conditions to obtain [1]. Further weighing 5 g of purified water, 0.06 g of lactic acid and 0.1 g of sodium carboxymethylcellulose were dissolved in the water to obtain [2]. [2] was added to [1] and mixed thoroughly under vacuum conditions to obtain a drug-containing cream by crosslinking reaction. The drug-containing cream was coated (so that the thickness of the drug-containing cream was approximately 1.0 mm), cut to the specifications of conventional gels, and the amount of drug contained in each gel was set to 0.02 g. The gels were then dried and packaged.

[0033] Example 16 Preparation of ointment 40g of stearyl alcohol and 45g of white petrolatum were dissolved in a water bath and heated to 75°C. 1.46g of sodium dodecyl sulfate, 0.025g of hydroxybenzyl ester, 0.015g of hydroxybenzyl propyl ester, 13g of propylene glycol, and 0.2g of polypeptide TRF7 were sequentially dissolved in water and heated to 75°C. Then, stearyl alcohol and white petrolatum at 75°C were added, and the mixture was stirred until cooled to obtain an ointment containing 0.2% TRF7.

[0034] Example 17 Preparation of liniment 0.1 g of polypeptide TRF7 was taken, dissolved in 45 mL of distilled water, filtered, and 5 mL of glycerol and 1 mL of nitrogen ketone were added to the filtrate. Distilled water was then added until the volume reached 50 mL to obtain a liniment containing 0.2% TRF7.

[0035] Example 18 Preparation of adhesive bandage 2 g of polyhydroxy 40 stearate, 2 g of polysorbate 80, 1 g of poloxamer 188, 0.3 g of sodium dihydrogen phosphate, 0.1 g of ethyl hydroxybenzene, and 50 mL of purified water were weighed, sterilized by heating at 130°C for 30-60 minutes, cooled to 30-40°C, and mixed uniformly to obtain [1]. 10 g of light liquid paraffin, 0.5 g of stearyl alcohol, 0.5 g of stearyl alcohol, 3 g of white petrolatum, and 1 g of glyceryl monostearate were weighed, sterilized, and kept warm at 30-40°C to obtain [2]. 0.1 g of polypeptide TRF7 was dissolved in 50 mL of distilled water, filtered, and added to [1], and mixed to obtain [3]. [2] was slowly added to [3], 3g of 15% ethanol was added, and the mixture was rapidly stirred to adjust the pH to 6.0. The temperature was maintained at 45°C, and the mixture was rapidly sheared and emulsified for 5 minutes. The mixture was stirred and cooled to 30-35°C to prepare a white, homogeneous cream liquid. The cream liquid was poured into a charging plate and coated onto the inner pads using a coating machine. The pads were then dried at a low temperature (45°C), cut to the required size, and obtained chemically coated inner pads. The coated inner pads were bonded to the center of the base fabric, and release paper was attached to the bonded ends of the base fabric.

[0036] Example 19 Preparation of facial cream Prepared 0.1g of polypeptide TRF7, mixed with 1g of No.26 white oil, 1g of stearyl alcohol, 1g of stearic acid, 0.5g of monoglycerin, 0.05g of 350 silicone oil, 0.5g of GTCC, and 0.03g of methylnipadin, and heated to 90°C to prepare the first semi-finish. To the first semi-finish, 4g of deionized water, 0.1g of polypeptide TRF7, 0.3g of pimelic acid O-20, and 0.5g of glycerol were added and heated to 80°C to prepare the second semi-finish. The second semi-finish was homogenized twice at 80°C (rotation speed 3000 rpm, time 10 minutes), then stirred for 30 minutes, cooled to 45°C, and after becoming creamy, 0.005g of cazon was added and mixed uniformly to prepare 1% polypeptide-containing creams.

[0037] Example 20 Preparation of a facial mask 0.1 g of polypeptide TRF7 was dissolved in 10 mL of deionized water, filtered, and the filtrate was mixed with 0.3 g of carbomer moistened with glycerin. Triethanolamine was added to adjust the pH to 6-7, and the mixture was uniformly spread onto mask paper to prepare masks containing 0.01 g of polypeptide per mask.

[0038] Example 21 Preparation of lotion 0.1 g of polypeptide TRF7 was taken and prepared as a 0.2 mg / mL aqueous solution. 1 g of hyaluronic acid was dispersed in 5 g of glycerol, dissolved in water, and thoroughly stirred to obtain a hyaluronic acid solution. 1 g of trehalose and 1 g of allantoin were dissolved in water and mixed with the above hyaluronic acid solution, and thoroughly stirred to obtain a mixed solution. The polypeptide solution, 1 g of D-panthenol, 10 g of oat β-glucan, 10 g of 1,2-pentanediol, and 0.05 g of preservative were sequentially added to the above mixed solution, and water was added and thoroughly stirred to obtain the final solution.

[0039] Example 22: Preparation of lotion (1) 0.1 g of polypeptide TRF7 was prepared as an aqueous solution at a concentration of 0.2 mg / mL. (2) 0.05 g of sodium hyaluronate was dissolved and dispersed in water, and the mixture was stirred well to obtain a hyaluronic acid solution. (3) 5g of glycerol, 3g of butanediol, 2g of trehalose, 0.2g of allantoin, and 0.1g of thickener were dissolved in water and heated to 75°C. (4) 2 g of jojoba seed oil, 3 g of hydrogenated polydecene, 2 g of polydimethylsiloxane, 3 g of caprylic / capric triglyceride, and 3 g of emulsifier were heated to 75°C and stirred uniformly. (5) The mixture prepared in step (3) was quickly poured over the mixture prepared in step (4), homogenized at constant temperature for 3-5 minutes, and then cooled. (6) Cool to below 60°C, add (1) and (2) and homogenize, cool to below 40°C, add preservative and fragrance to obtain lotion.

[0040] Example 23: Preparation of cleansing gel 0.1 g of polypeptide TRF7 was prepared as a 0.2 mg / mL aqueous solution. The polypeptide aqueous solution, 2.5 g of glycerol, 3 g of decyl glucoside, 3 g of sodium cocoyl apple amino acid, 1.5 g of sodium laureth sulfate, 0.1 g of solubilizer, and 0.05 g of fragrance were sequentially added to water and stirred. Then, 0.5 g of thickener was added and stirred until completely dissolved to obtain a cleansing gel.

[0041] Example 24: Evaluation of the cosmetic effects of a novel polypeptide lotion. 1.1 Test sample: Polypeptide lotion prepared in Example 22 of the present invention 1.2 Participants: 30 individuals in total, 18 women and 12 men aged 18 to 55. The participants had unsightly skin conditions such as wrinkles, hyperpigmentation, dullness, and scars from minimally invasive surgery or post-surgical procedures. 1.3 Test Method: After cleansing the subject's skin, the lotion prepared in Example 22 is applied to the skin, and the effects of use are observed and felt. 1.4 The results of the test evaluation are shown in Table 6. JPEG0007855811000008.jpg55164 The subjects reported that using the novel polypeptide TRF7 lotion product increased skin moisture content, improved skin hydration and elasticity, and enhanced skin moisture and hydration. The product also showed whitening effects and effects in removing wrinkles, scars, and hyperpigmentation. No allergies were observed in the subjects.

[0042] Example 25: Evaluation of the cosmetic effects of a novel polypeptide mask. 1.1 Test sample: A facial mask prepared according to Example 20 of the present invention. 1.2 Participants: 30 individuals in total, 21 women and 9 men, aged 18 to 58. The facial skin of the participants had unsightly factors such as wrinkles, hyperpigmentation, dullness, and scars from minimally invasive surgery or post-surgical procedures. 1.3 Test Method: After washing the subjects' faces, the novel polypeptide mask prepared in Example 20 was applied to their faces once a day, and the effects of use were observed and felt. 1.4 The results of the test evaluation are shown in Table 7. JPEG0007855811000009.jpg55164 The subjects reported that using the novel polypeptide TRF7 mask product increased skin moisture, improved skin hydration and elasticity, and enhanced skin moisture and hydration. The product also showed whitening effects and effects in removing wrinkles, scars, and hyperpigmentation. No allergies were observed in the subjects.

[0043] The above embodiments are preferred embodiments of the present invention, but embodiments of the present invention are based on the above embodiments. Therefore, it is not limited to the spirit and principles of the present invention, and can be carried out without departing from them. Any other changes, modifications, substitutions, combinations, and simplifications are equivalent substitutions and all are protected by the present invention. It shall be included in the scope. (Note) (Note 1) A novel polypeptide for promoting tissue repair, The novel polypeptide is characterized by having the following structure: JPEG0007855811000010.jpg31170 Here, R 1 =H, Ala, Gly, Ala-Ala- or Val-Ala-, R 2 =Asn or Asp, R 3 =Ser, Tyr, -Gly-Ser-Tyr, -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr, -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His -Val-Arg, or -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His-Val-Arg-Glu-Tyr-Pro-Ala-Gly-Val-Ser-Ala-Ala. (Note 2) The novel polypeptide according to Appendix 1, characterized in that the novel polypeptide has a structure represented by any one of TRF1 to TRF12. JPEG0007855811000011.jpg100170 (Note 3) Use of the novel polypeptides described in Appendix 1 or 2 in the preparation of products for promoting tissue repair or improving skin aesthetics. (Note 4) The aforementioned product is a pharmaceutical, medical device, or daily chemical product, and is used in the preparation of a novel polypeptide described in Appendix 3 for promoting tissue repair or improving skin aesthetics. (Note 5) The aforementioned pharmaceuticals, medical devices, or daily chemical products include one or more novel polypeptides in an effective amount, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, with the remainder being an additive or other compoundable agent, and are used in the preparation of products for promoting tissue repair or improving skin aesthetics as described in Appendix 4. (Note 6) The aforementioned pharmaceuticals, medical devices, or daily chemical products are tablets, capsules, injections, liposome nanoparticles, release control agents, gel creams, ointments, liniments, patches, creams, cleansers, lotions, gels, or toners, and are used in the preparation of products for promoting tissue repair or improving skin appearance as described in Appendix 4. (Note 7) The aforementioned additive is a solvent, disintegrant, flavor enhancer, preservative, colorant, or binder, and is used in the preparation of products for promoting tissue repair or improving skin appearance of the novel polypeptide described in Appendix 5. (Note 8) A pharmaceutical composition characterized by containing a novel polypeptide described in Appendix 1 or 2 and a pharmaceutically acceptable carrier.

Claims

1. A novel polypeptide for promoting tissue repair, The novel polypeptide is characterized in that it has the structure shown in any one of TRF1 to TRF12.

2. Use of the novel polypeptide described in claim 1 in the preparation of products for promoting tissue repair or improving skin aesthetics.

3. The aforementioned product is a pharmaceutical, medical device, or daily chemical product, and is used in the preparation of a novel polypeptide according to claim 2 for promoting tissue repair or improving skin aesthetics.

4. The use of the novel polypeptide according to claim 3 in the preparation of a product for promoting tissue repair or improving skin appearance, wherein the aforementioned pharmaceutical, medical device, or daily chemical product comprises one or more novel polypeptides in an effective amount, or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, with the remainder being an additive or other compoundable agent.

5. The use of the novel polypeptide according to claim 3 in the preparation of products for promoting tissue repair or improving skin appearance, wherein the pharmaceutical product, medical device, or daily chemical product is a tablet, capsule, injection, liposome nanoparticle, release control agent, gel cream, ointment, liniment, patch, cream, cleanser, lotion, gel, or toner.

6. The use of the novel polypeptide according to claim 4 in the preparation of a product for promoting tissue repair or improving skin appearance, wherein the additive is a solvent, a disintegrant, a flavor enhancer, a preservative, a colorant, or a binder.

7. A pharmaceutical composition characterized by containing a novel polypeptide as described in claim 1 and a pharmaceutically acceptable carrier.

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