Application of compound in preparation of scar relieving agent and external scar removing agent

Abstract

The invention belongs to the technical field of skin wound repair, and particularly relates to application of a compound in preparation of a scar relieving agent and an external scar removing agent. According to anti-scar traditional Chinese medicine prescription analysis and component and activity research, daucosterol, shikonin, przewalskin C and 4-ethoxyformyl-quinol-2-ketone have the effects of inhibiting proliferation of human keloid fibroblasts and inhibiting collagen synthesis of human normal skin fibroblasts induced by TGF-beta1; the composition has a scar relieving effect on scars of scar animal models, and can be used as a scar relieving agent, especially an external preparation applied to local skin. The invention further discloses an external scar-removing agent, namely an emulsion-type liniment, the main component of the external scar-removing agent contains silicone oil and also contains any one of an antioxidant and / or daucosterol, shikonin, przewalskin C and 4-ethoxyformyl-quinoline-2-ketone, and the main component has a better effect of relieving scars of a scar animal model when being externally applied to local skin.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin wound repair, and specifically relates to the application of a compound in the preparation of a scar-reducing agent and an external scar-removing agent. Background Art

[0002] Scar formation is an inevitable result of wound healing. However, the morphology of the skin repaired compensatorily is often different from that of the original skin. During the wound repair process, excessive proliferation of skin fibroblasts and excessive deposition of collagen easily lead to pathological scars, such as hypertrophic scars and keloids, which affect the appearance and even the skin function.

[0003] Physical means such as surgery and laser have been used to reduce scars, but the use of drugs or cosmetics is more convenient and is currently the main means of reducing skin scars. "Current Status of Drug Therapy for Scars" published by Jiang Jiang et al. in the 1st issue of Volume 36 of the Journal of Clinical Military Surgeons in 2008 introduced commonly used scar-reducing agents, also known as scar lightening agents, scar-removing agents or scar-eradicating agents, including glucocorticoids, all-trans retinoic acid, pingyangmycin, silicone (silicone oil) and Chinese herbal extracts or monomeric compounds in Chinese herbs. Among them, scar-reducing agents such as glucocorticoids and pingyangmycin that need to be injected have the problems of inconvenient use and relatively large side effects.

[0004] In recent years, it has been realized at home and abroad that traditional Chinese medicine has the potential in reducing scars, and certain progress has been made in the research. Reviews such as "Research Progress in Drug Therapy for Pathological Scars" published by Li Guifeng et al. in the 9th issue of Volume 23 of Chinese Journal of Aesthetic Medicine in 2014 have been carried out on this. Traditional Chinese medicine such as Zihua Burn Ointment, Scald Ointment, Compound Scar Cream, Scar No. 1, Scar Ointment, etc. have shown good scar-reducing effects in clinical applications. Onion extract has the effect of reducing scars, and flavonoids such as quercetin and kaempferol in it are considered to be the active ingredients for reducing scars, and onion extract is widely used in related patents such as CN107496661A. Monomeric substances of traditional Chinese medicine for removing scars that have been deeply studied include tanshinone IIA, asiaticoside, glycyrrhizin, ginsenoside Rg3, oleic acid, linoleic acid, sesamin, curcumin, hyperin, shikonin, etc. The listed Meibao Scar Ointment uses oleic acid, linoleic acid, etc. as the main ingredients. Shikonin, asiaticoside, etc. are more applied in related patents such as CN109620859A. The above substances usually regulate the proliferation of skin fibroblasts and collagen deposition through signal pathways such as TGF-β, PI3K, and retinoic acid receptors, thereby reducing scars. In addition, heat shock protein Hsp90, etc. also participate in the regulation of skin fibroblast proliferation and collagen synthesis. Reviews such as "Research Progress in the Relationship between Heat Shock Protein and Pathological Scar Formation" published by Tang Xiaoyan et al. in the 4th issue of Volume 48 of New Medicine in 2017 have been carried out on this.

[0005] Due to the side effects of Western medicines for injection and the inconvenience of administration, traditional Chinese medicines and monomer compound anti-scar products have problems such as more ingredients and complex compatibility. New scar-reducing agents and anti-scar topical agents are still hotspots in the fields of medicine and cosmetics. Summary of the Invention

[0006] In view of the above-mentioned current situation and existing problems of scar-reducing agents and anti-scar topical agents, the present invention analyzes and studies the formula rules, ingredients, etc. of clinical anti-scar traditional Chinese medicine compounds, and discovers several new scar-reducing compounds that have regulatory effects on the proliferation of skin fibroblasts and collagen synthesis, and develops a convenient-to-use anti-scar topical agent to be used as a medicine or cosmetic for preventing or reducing scars, such as hypertrophic scars or keloids in pathological scars.

[0007] Therefore, the present invention first provides the use of a compound in the preparation of a scar-reducing agent, and the compound includes daucosterol, arnebinol, przewaquinone c, and 4-ethoxycarbonyl-2-quinolone. Among them, daucosterol: molecular formula C 35 H 60 O6, molecular weight 576.8 g / mol, CAS: 474-58-8, exists in traditional Chinese medicines such as Prunella vulgaris, Brucea javanica, and Gleditsia sinensis. Arnebinol (also known as neoarnebinol, arnebifoliol; Arnebinol) molecular formula C 16 H 20 O2, molecular weight 244.33 g / mol, CAS: 87064-17-3, exists in traditional Chinese medicines such as Arnebia euchroma. Przewaquinone c (also known as przewalskin C; Przewaquinone c): molecular formula C 18 H 16 O4, molecular weight 296.3 g / mol, CAS: 189290-30-0, exists in the traditional Chinese medicine Salvia miltiorrhiza. 4-Ethoxycarbonyl-2-quinolone: molecular formula: C 12 H 11 NO3, molecular weight 217.22 g / mol, CAS: 5466-27-3, exists in traditional Chinese medicines such as Brucea javanica.

[0008] Studies on the above compounds in vitro and by topical skin application have shown that they have the effects of inhibiting the proliferation of human keloid fibroblasts, inhibiting the collagen synthesis of human normal skin fibroblasts induced by TGF-β1, inhibiting scar hyperplasia in a rabbit ear hypertrophic scar model, and reducing scars and having a certain promoting effect on wound healing in rabbit contusion wounds and incision suture wounds. Therefore, it is suitable for topical skin application, including local injection or topical application.

[0009] Preferably, the scar reducing agent is an externally applied preparation for topical application to the skin, including but not limited to externally applied tinctures, ointments, creams, pastes, aerosols, sprays, lotions, liniments, paints, film-forming agents, gels, and patches.

[0010] The present invention further studies and discovers that taking silicone oil and an antioxidant as the main components; or taking silicone oil added with any one of daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one as the main component; or taking silicone oil, antioxidant added with any one of daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one as the main component has a good inhibitory effect on scar hyperplasia in a rabbit ear hypertrophic scar model.

[0011] The present invention further improves the externally applied scar-removing preparation and invents an emulsion film-forming agent. The main components of the externally applied scar-removing agent are: taking silicone oil and an antioxidant as the main components; or taking silicone oil added with any one of daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one as the main component; or taking silicone oil, antioxidant added with any one of daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one as the main component. The externally applied scar-removing agent further contains a film-forming material, a penetration enhancer, an emulsifier, and a humectant.

[0012] Among them, the film-forming material can be selected from at least one of polyvinyl alcohol (including but not limited to PVA0588, PVA1788, PVA1799), sodium alginate, and hydroxypropyl methylcellulose; the penetration enhancer can be selected from at least one of menthol, borneol, wintergreen oil, and azone; the emulsifier can be selected from at least one of Peregal (including but not limited to O-25, O-15, O-9), poloxamer (including but not limited to poloxamer 188, poloxamer 407), Tween (including but not limited to Tween-80, Tween-60, Tween-20), and cetyl alcohol; the humectant can be selected from at least one of allantoin, propylene glycol, glycerol, sodium polyglutamate, sodium hyaluronate, and urea; the antioxidant can be selected from at least one of gallic acid, tea polyphenols, gallotannic acid, paeonol, vitamin C, and vitamin E.

[0013] The preferred antioxidant is one of gallic acid, gallotannic acid, and paeonol.

[0014] The preferred molecular weight of sodium polyglutamate is 20,000 - 30,000 Da.

[0015] The preferred average molecular weight of sodium hyaluronate is 50,000 - 700,000 Da.

[0016] The preferred degree of polymerization of sodium alginate is 80 - 750.

[0017] The viscosity range of the preferred 2% aqueous solution of hydroxypropyl methylcellulose at 20 °C is 400 to 200,000 mPa·s.

[0018] The average molecular weight of the preferred silicone oil is 500 to 6,000; the silicone oil is an unmodified silicone oil.

[0019] Taking silicone oil as the standard, as a typical prescription of an external scar-removing agent, the weight ratio of silicone oil to film-forming material, penetration enhancer, emulsifier, and humectant in the external scar-removing agent is: silicone oil 0.5 to 10 parts; film-forming material 10 to 40 parts; emulsifier 1 to 40 parts; humectant 5 to 40 parts; penetration enhancer 0.1 to 2 parts. The weight ratio of silicone oil to film-forming material, penetration enhancer, emulsifier, and humectant in the preferred external scar-removing agent is: silicone oil 2 to 10 parts; film-forming material 22.5 to 32.5 parts; emulsifier 19 to 32 parts; humectant 15 to 30 parts; penetration enhancer 0.5 to 1 part.

[0020] Taking silicone oil as the standard, as a typical prescription of an external scar-removing agent, the weight ratio of silicone oil to antioxidant in the external scar-removing agent is: silicone oil 0.5 to 10 parts; antioxidant 0.5 to 2 parts. The weight ratio of silicone oil to antioxidant in the preferred external scar-removing agent is: silicone oil 2 to 10 parts; antioxidant 0.5 to 0.75 part. In addition to containing film-forming material, penetration enhancer, emulsifier, and humectant, this external scar-removing agent can be mainly composed of silicone oil and antioxidant; or mainly composed of silicone oil, antioxidant, and any one of daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one. The weight ratio of silicone oil to film-forming material, penetration enhancer, emulsifier, and humectant in this external scar-removing agent can adopt the aforementioned ratio.

[0021] Taking silicone oil as the standard, as a typical prescription of an external scar-removing agent, the weight ratio of silicone oil to daucosterol, shikonol, salvianolic acid C or 4-ethoxycarbonyl-quinolin-2-one in the external scar-removing agent is: silicone oil 0.5-10 parts; daucosterol 0.1-4.5 parts; shikonol 0.1-1.8 parts; salvianolic acid C 0.1-2.7 parts; 4-ethoxycarbonyl-quinolin-2-one 0.1-1.8 parts. The preferred weight ratio of silicone oil to daucosterol, shikonol, salvianolic acid C or 4-ethoxycarbonyl-quinolin-2-one in the external scar-removing agent is: silicone oil 2-10 parts; daucosterol 0.3-0.9 parts; shikonol 0.12-0.36 parts; salvianolic acid C 0.18-0.54 parts; 4-ethoxycarbonyl-quinolin-2-one 0.12-0.36 parts. In addition to film-forming materials, penetration enhancers, emulsifiers, and moisturizers, the external scar-removing agent can use any one of daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one added with silicone oil as the main component; or use any one of daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one added with silicone oil and antioxidants as the main component. The weight ratio of silicone oil to film-forming materials, penetration enhancers, emulsifiers, and moisturizers in the external scar-removing agent can adopt the aforementioned ratio.

[0022] The scar-reducing agent and the external scar-removing preparation of the present invention do not belong to antibiotics, and it is preferably used after the epithelialization of the skin wound, which can reduce the irritation to the open skin wound.

[0023] The present invention further investigated the preparation method of the external scar-removing preparation, and invented a preparation method of the aforementioned external scar-removing preparation, including the following steps: Take purified water, add a moisturizer, a film-forming material and the main component except silicone oil according to weight parts, and stir evenly to prepare an aqueous phase; Take silicone oil, an emulsifier, and a penetration enhancer according to weight parts and put them into an emulsifying machine, heat and dissolve to prepare an oil phase; Vacuum suck the aqueous phase into the emulsifying machine, homogenize and emulsify, and cool the interlayer of the emulsifying machine by passing cold water, and then fill. Beneficial effects

[0024] Daucosterol, shikonol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one of the present invention have the effects of inhibiting the proliferation of human keloid fibroblasts and inhibiting the collagen synthesis of human normal skin fibroblasts induced by TGF-β1, have an inhibitory effect on scar hyperplasia in a rabbit ear hypertrophic scar model, and have a scar-reducing and certain wound-healing promoting effect on rabbit contusion wounds and incision suture wounds; and the multi-target action is beneficial to reducing the components of the prescription.

[0025] In the scar-removing emulsion-type film-forming agent of the present invention, daucosterol, alkannol, salvianolic acid C, and 4-ethoxycarbonyl-quinolin-2-one have good effects in inhibiting scar hyperplasia; silicone oil has a certain effect in inhibiting scar hyperplasia and can soften scars; long-term use of antioxidants has the effect of fading scar pigmentation; humectants protect the wound and increase the hydration environment on the scar surface; penetration enhancers improve the penetration of the main components into the stratum corneum and increase their accumulation in scar tissues. The main components and excipients cooperate with each other, which is beneficial to improving the improvement effect of the film-forming agent on scars.

[0026] The emulsion-type film-forming agent of the present invention can form a uniform, transparent, beautiful, and flexible film after drying at the scar or wound site, which can be peeled off, does not stain clothes, and is convenient to use. Brief Description of the Drawings

[0027] Figure 1 is the optical density value at 570 nm of each group of cells determined by the methyl thiazolyl tetrazolium colorimetric method after treating human keloid fibroblasts with the test compound; Figure 2 is the determination result of the soluble and insoluble collagen contents of each group of cells after treating human normal skin fibroblasts with the test compound; Figure 3 is the hematoxylin and eosin staining map of typical pathological sections of each group of rabbit ears in the rabbit ear hypertrophic scar model treated with the test substance; Figure 4 is the scar elevation index result of some treatment groups after treating the rabbit ear hypertrophic scar model with the test substance; Figure 5 is the skin healing and scar conditions of the contusion wounds of rabbits and 2 linear sutured incision wounds on the 3rd and 7th days after ENQ administration; In the figure, DMSO is dimethyl sulfoxide, MDS is asiaticoside, SKN is shikonin, STL is daucosterol, ANB is alkannol, PWQ is salvianolic acid C, and ENQ is 4-ethoxycarbonyl-quinolin-2-one. Detailed Embodiments

[0028] The following clearly and completely elaborates on the technology of the present invention through some typical embodiments. The silicone oil used in the embodiments is non-modified silicone oil.

[0029] For the sake of brevity, the following chemical substances, cells, proteins / receptors are represented by unified codes: (1) Dimethyl sulfoxide: DMSO; (2) Sitosterol: STL; (3) Arnebinol: ANB; (4) Salvianolic acid C: PWQ; (5) 4-Ethoxycarbonyl-2-quinolone: ENQ; (6) Asiaticoside: MDS; (7) Shikonin: SKN; (8) Human keloid fibroblasts: HKFs; (9) Human normal skin fibroblasts: HDFs; (10) Heat shock protein 90: HSP90; (11) Retinoic acid receptor: RAR; (12) TGF-β receptor 1: TβR1.

[0030] Example 1 Regulation of HKFs Proliferation by Compounds 1.1 Compounds and Solvents Test compounds: MDS (positive control), STL, ANB, PWQ, ENQ; Compound solvent: DMSO. The test compound solutions were prepared freshly before use.

[0031] 1.2 Cell Culture and Cell Proliferation Test HKFs (ATCC CRL-1762) were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, penicillin (30 U / mL), and streptomycin (100 μg / mL). After resuscitation and subculture, the 4th generation cells were used for the drug addition test. HKFs were added with test compounds (concentration gradients of STL, ANB, PWQ, ENQ: 4 μM, 20 μM, 100 μM; MDS concentration: 100 μM) and cultured in a CO2 incubator for 96 h (37°C, 5% CO2).

[0032] After the drug addition culture was completed, HKFs were diluted with fresh Dulbecco's modified Eagle's medium and inoculated into a cell culture plate at 1x10 5 cells / well. 20 μL (5 mg / mL) of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and cultured at 37°C and 5% CO2 for 4 h. The supernatant was discarded, and 200 μL of DMSO was added to dissolve MTT. The optical density value (OD value) at 570 nm was measured to evaluate cell proliferation.

[0033] 1.3 Regulation of HKFs Proliferation by Compounds After adding each test compound, the OD value at 570 nm was measured by the MTT colorimetric method. As Figure 1As shown, after 96 hours of MDS, STL, ANB, PWQ, and ENQ treatment, the OD values were all lower than those of DMSO (t-test, p < 0.05). Among them, after 96 hours of treatment with STL 100 μM, ANB 100 μM, PWQ 100 μM, ENQ 20 μM, and ENQ 100 μM, the OD values were all lower than those of the MDS 100 μM treatment group (t-test, p < 0.05). STL, ANB, PWQ, and ENQ have an inhibitory effect on the proliferation of HKFs, and the inhibitory effect is higher than that of MDS at the same concentration and has a certain concentration correlation.

[0034] Example 2 Regulation of Collagen Synthesis in HDFs by Compounds 2.1 Compounds and Solvents Test compounds: SKN (positive control), STL, ANB, PWQ, ENQ; Compound solvent: DMSO. The test compound solutions were prepared freshly before use.

[0035] TGF-β1 was dissolved in 0.1% bovine serum albumin solution (w / v) containing 4 mM HCl for standby.

[0036] 1.2 Cell Culture and Collagen Synthesis Determination HDFs (ATCC PCS-201-012) were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, penicillin (30 U / mL), and streptomycin (300 μg / mL). After resuscitation and subculture, the 4th generation cells were used for drug addition tests.

[0037] DMSO (blank control) and TGF-β1 (model control, 10 ng / ml) were added to HDFs; in addition, test compounds were added to HDFs treated with TGF-β1 (concentration gradients of STL, ANB, PWQ, ENQ: 2 μM, 10 μM, 50 μM; SKN concentration: 2 μM). After drug addition, the HDFs were cultured in a CO2 incubator for 72 hours (37°C, 5% CO2).

[0038] After the drug addition culture was completed, the soluble and insoluble collagens in the culture medium and cells were measured using the Sircol Collagen Assay Kit according to the operation instructions.

[0039] 1.3 Effects of Compounds on Collagen Synthesis in HDFs After adding each test compound, the measurement results of soluble and insoluble collagens in the culture medium and cells are respectively as Figure 2 A and Figure 2As shown in B. TGF-β1 can induce an increase in the content of soluble and insoluble collagen in HDFs (t-test, p < 0.05). SKN, STL, ANB, PWQ, and ENQ can all reduce the increase in the content of soluble and insoluble collagen induced by TGF-β1 (t-test, p < 0.05). The content of soluble collagen in each concentration group of STL, the 10 μM and 50 μM concentration groups of ANB, the 10 μM and 50 μM concentration groups of PWQ, and each concentration group of ENQ is lower than that in the 2 μM group of SKN (t-test, p < 0.05); there is no significant difference in the content of soluble collagen between the 2 μM groups of ANB and PWQ and the 2 μM group of SKN (t-test, p > 0.05). The content of insoluble collagen in each concentration group of STL, ANB, PWQ, and ENQ is lower than that in the 2 μM group of SKN (t-test, p < 0.05). STL, ANB, PWQ, and ENQ have an inhibitory effect on the increase in collagen synthesis in HDFs induced by TGF-β1, showing a certain concentration correlation.

[0040] Example 3 Protein / Receptor Docking Test of Compounds This example demonstrates the methods and results of the protein / receptor docking test for some compounds during the research process. The selected protein / receptor is related to the regulation of fibroblast proliferation and collagen synthesis.

[0041] 3.1 Method 1 Reference for Method 1: 《Synthesis, docking, and in vitro studies of somesubstituted bischalcones on acid and alkaline phosphatases》 (Authors Singh M, Raghav N; Journal Volume and Page Numbers 《Medicinal Chemistry Research》 2014, 23:1781 - 1788).

[0042] 3.2 Test Results and Conclusions of Method 1 The proteins / receptors of Method 1 include HSP90, RAR, and TβR1; the compounds include STL, ANB, PWQ, and ENQ. The main test results are shown in Table 1 (values are retained to 1 decimal place).

[0043] Table 1 Test Results of Method 1

[0044] The binding energies of the four compounds to HSP90, RAR, and TβR1 (approximately the sum of the van der Waals force and hydrogen bond energies) are all lower than -89, which are similar to or lower than those of the corresponding proteins / receptors of the known HSP90 small molecule inhibitor 17 - acrylamino - 17 - demethoxygeldanamycin and its derivatives, and the RAR small molecule agonist all - trans retinoic acid, indicating good binding stability. Among them, the intermolecular van der Waals force is negatively correlated with the molecular weight of the compound. The intermolecular hydrogen bond energy between the protein / receptor and the four compounds is the lowest for ENQ and the highest for ANB (the intermolecular hydrogen bond energy between 1YET and 3TZM and PWQ is the highest; the intermolecular hydrogen bond energy between 2LBD and STL is the highest).

[0045] 3.4 Supplementary tests 3.4.1 Method 2 Reference for Method 2: 《Possible SARS - coronavirus 2 inhibitor revealed by simulated molecular docking to viral main protease and host toll - like receptor》 (authors Hu X, Cai X, etc.; journal volume and page numbers 《Future Virology》, 2020, 15(1):359 - 368.).

[0046] 3.4.2 Test results and conclusions of Method 2 The proteins / receptors of Method 2 include HSP90, RAR, TβR1 (PDB IDs are shown in Table 1); the compounds include STL, ANB, PWQ, and ENQ. The binding energies of the four compounds to HSP90, RAR, and TβR1 are all lower than -5 kcal / mol, and the lowest binding energy is -10.18 kcal / mol, showing good binding effects.

[0047] Combined with Examples 1 and 2, the above results indicate that the compounds STL, ANB, PWQ, and ENQ can regulate the proliferation and collagen synthesis activity of skin fibroblasts by targeting HSP90, RAR, and TβR1.

[0048] Example 4 Investigation of rabbit ear hypertrophic scar animal model This example demonstrates the effects of the compounds investigated during the research on hypertrophic scars.

[0049] 4.1 Experimental animals and test drugs Male New Zealand white rabbits (2.5 kg - 3.0 kg). After a 3 - day pre - examination, they were raised in a conventional environment.

[0050] Control and test drugs: The controls and test drugs set are shown in Table 2. For the test drugs containing silicone oil, such as the test drug of gallic acid (GA) and silicone oil, silicone oil is administered alone for dosing; GA uses DMSO as the solvent and is freshly prepared and administered alone. The treatment of other test drugs containing silicone oil is the same. DMSO is the excipient control, and GA + silicone oil is the positive control.

[0051] Table 2 Settings, solvents and dosages of controls and test drugs

[0052] Note 1: GA is gallic acid, and the molecular weight of silicone oil is 1000; Note 2: In the volume dosage, for the test drugs containing silicone oil, it is calculated based on the total volume of DMSO and silicone oil; Note 3: In the test drug dosage, for the test drugs containing silicone oil, the silicone oil dosage is 100 mg / kg / d, and the dosage in the table is the dosage of the component other than silicone oil in the test drug.

[0053] 4.2 Methods 4.2.1 Model making Method reference: "Establishment of a rabbit ear hypertrophic scar model" (authors Niu Fuyou, Niu Yonggan, Chen Yantang; journal volume and page numbers "China Medical Engineering", 2004, 12(5):4). The model was established according to the method of making a rabbit ear hypertrophic scar model. New Zealand white rabbits were anesthetized with pentobarbital, the ears were depilated and routinely disinfected, and punctures were made on the ventral surface of one ear to make 5 circular wounds with a diameter of 10 mm reaching the cartilage. After the wounds were established, the wound healing was observed weekly, and after the wounds showed hypertrophic lesions, the animals were randomly grouped for drug administration.

[0054] 4.2.2 Grouping, drug administration and observation The New Zealand white rabbits used for model making were randomly divided into 18 groups, with 2 rabbits in each group and 10 wounds. They were self-controlled on the left and right (the ear with the wound was the model side; the ear without the wound was the normal control side); the ears on the model side of each group were respectively dropped and smeared with the controls and test drugs in 4.1 according to the dosages in Table 2. Each animal was smeared twice a day (with an interval of 4 - 5 hours) for 3 consecutive weeks, and the local recovery of the scar was observed weekly.

[0055] After 3 weeks of drug administration, the animals were sacrificed, and samples of the wound area, the surrounding ear and the normal ear were taken, fixed with 10% formalin, embedded in paraffin to make 5-μm sections, stained with hematoxylin and eosin, and subjected to pathological observation. The scar elevation index (SEI) was measured under a 10-fold microscope = the height difference from the highest point on the scar surface to the cartilage surface / the height difference from the normal skin surface around the scar to the cartilage surface.

[0056] 4.3 Results After 3 weeks of drug administration, typical pathological section staining diagrams of the normal ear and the modeled wound ear are shown in Figure 3. After modeling, the SEI of the adjuvant control group was significantly higher than that of the normal control ear (t-test, p<0.05). The SEI of the positive control group, the medium and high dose groups of STL, ANB, PWQ, and ENQ, and the medium and high dose groups of STL+silicone oil, ANB+silicone oil, PWQ+silicone oil, and ENQ+silicone oil was lower than that of the adjuvant control (t-test, p<0.05), with fewer new blood vessels and cell infiltration areas in the scar and regular cell arrangement.

[0057] Among them, the SEI of the high dose groups of STL, ANB, PWQ, and ENQ, and the STL+silicone oil, ANB+silicone oil, PWQ+silicone oil, and ENQ+silicone oil groups was lower than that of the positive control group (t-test, p<0.05). The SEI of the STL+silicone oil, ANB+silicone oil, PWQ+silicone oil, and ENQ+silicone oil groups was lower than that of the high dose groups of STL, ANB, PWQ, and ENQ respectively, although the difference was not significant (t-test, p>0.05). The SEI of the low dose groups of STL, ANB, PWQ, and ANB was lower than that of the adjuvant control, and the difference was not significant (t-test, p>0.05). Figure 4 Shows the scar SEI comparison of each modeling group with SEI lower than that of the adjuvant control.

[0058] Example 5 Investigation of the healing of contusion wounds and incision suture wounds in rabbits and safety This example shows the effects and safety of the compounds investigated during the research on the healing of contusion wounds and incision suture wounds.

[0059] 5.1 Experimental animals and test drugs Experimental animals: Male New Zealand white rabbits (2.5 kg - 3.0 kg). After a 3-day pre-examination, they were raised in a conventional environment.

[0060] Control and test drugs: The blank adjuvant control was DMSO; the drug-loaded test drugs included: DMSO+STL (STL dose 225 mg / kg / d), DMSO+ANB (ANB dose 90 mg / kg / d), DMSO+PWQ (PWQ dose 135 mg / kg / d), DMSO+ENQ (ENQ dose 90 mg / kg / d).

[0061] 5.2 Methods 5.2.1 Model making Eight rabbits, 2 for each test drug, had their left and right dorsal skin depilated with a depilatory. Two days after depilation, 1 circular contusion (i.e., abrasion) wound with equal area or length and 2 linear incision suture wounds were made on the left and right depilated parts of each rabbit.

[0062] 5.2.2 Grouping The experiment was a self - left - right control. The test drug was applied to the right side, and the blank excipient was applied to the left side. The test drug was applied externally for 7 days, and the dressing was changed once a day. After application, 4 layers of disinfected gauze were covered to prevent the test drug from being rubbed off during movement.

[0063] 5.2.3 Observation Indicators Observe the changes before treatment and 3 to 7 days after administration at the treatment and control sites, and record the allergic symptoms at the application site and the adverse reactions of the animals in the cardiovascular, respiratory, central nervous systems, etc. during the medication period and within 7 days after administration.

[0064] 5.3 Results During the medication period and within 7 days after administration, no obvious allergic symptoms were observed on the skin of rabbits. During the medication period and within 7 days after administration, the coordination ability of rabbits was normal, and no obvious adverse reactions were observed in the cardiovascular and respiratory systems, etc.

[0065] In the self - control, the healing of the contusion wound and the incision suture wound on the test drug side was better than that on the blank excipient side, and the scar was smaller than that on the blank excipient side; under the same conditions, the wound area or length on the test drug side was smaller than or approximately equal to that on the blank excipient side 3 to 7 days after administration. Among them, the contusion wound area and the incision suture wound length of the DMSO + ENQ test drug were smaller than those on the blank excipient side (see Figure 5 , the dark part is the wound site). The tested test drugs do not delay the healing of the contusion wound and the incision suture wound or are beneficial to accelerating the wound healing.

[0066] Example 6 Emulsion Coating Agent and Its Preparation Main component 1: 2 kg of silicone oil (molecular weight 1000); Main component 2: 0.3 kg of STL or 0.12 kg of ANB or 0.18 kg of PWQ or 0.12 kg of ENQ or 0.5 kg of GA; Emulsifier: 20 kg of Peregal O - 25, 5 kg of cetyl alcohol; Film - forming material: 30 kg of PVP0588, 2.5 kg of sodium alginate (degree of polymerization 600); Humectant: 15 kg of glycerol, 0.25 kg of urea, 0.25 kg of sodium polyglutamate (20000 Da); Penetration enhancer: 0.5 kg of menthol; Purified water was added to make 200 kg.

[0067] Preparation process: (1) Weigh purified water and add it to a blender. Stir at a speed of 15 rpm. At room temperature, add glycerol, urea, sodium polyglutamate, PVP0588, sodium alginate, and main component 2, and stir for 30 - 60 minutes until evenly mixed; (2) Stop the mixer, transfer the liquid material in step (1) to the dispensing tank, keep it warm at 72°C, stir at a speed of 20 rpm for 20 - 40 minutes, turn off the heating, seal the dispensing tank, and let it stand overnight to obtain the aqueous phase; (3) Add silicone oil, Peregal O - 25, and cetyl alcohol to the emulsifier, set the emulsification temperature at 72°C, add menthol before emulsification, and wait for 30 - 60 minutes until all the oil phase is dissolved and reaches 72°C; (4) Suck in the aqueous phase under vacuum, homogenize for 5 minutes, emulsify for 16 minutes, cool the liquid material, and fill it.

[0068] Example 7 Emulsion - type film - forming agent and its preparation Main component 1: Silicone oil (molecular weight 1800) 5 kg; Main component 2: STL 0.6 kg or ANB 0.24 kg or PWQ 0.36 kg or ENQ 0.24 kg or gallotannic acid 0.75 kg; Emulsifier: Peregal O - 9 15 kg, Tween - 80 1.5 kg, cetyl alcohol 5 kg; Film - forming material: PVP1788 25 kg; Humectant: Propylene glycol 15 kg, glycerol 15 kg; Penetration enhancer: Borneol 0.5 kg; Purified water is added to make 200 kg.

[0069] Preparation process: (1) Weigh purified water and add it to the mixer, stir at a speed of 15 rpm, add propylene glycol, glycerol, PVP1788, and main component 2 at room temperature, and stir for 30 - 60 minutes until well - mixed; (2) Stop the mixer, transfer the liquid material in step (1) to the dispensing tank, keep it warm at 75°C, stir at a speed of 20 rpm for 20 - 40 minutes, turn off the heating, seal the dispensing tank well, and let it stand overnight to obtain the aqueous phase; (3) Add silicone oil, Peregal O - 9, cetyl alcohol, and Tween - 80 to the emulsifier, set the emulsification temperature at 75°C, add borneol before emulsification, and wait for 30 - 60 minutes until all the oil phase is dissolved and reaches 75°C; (4) Suck in the aqueous phase under vacuum, homogenize for 8 minutes, emulsify for 15 minutes, cool the liquid material, and fill it.

[0070] Example 8 Emulsion - type film - forming agent and its preparation Main component 1: Silicone oil (molecular weight 2000) 5 kg; Main component 2: STL 0.6 kg or ANB 0.24 kg or PWQ 0.36 kg or ENQ 0.24 kg or tea polyphenols 0.75 kg; Emulsifier: Peregal O-25 10 kg, Peregal O-9 5 kg, cetyl alcohol 4 kg; Film-forming material: PVP1788 25 kg; Humectant: glycerol 25 kg, sodium polyglutamate (30000 Da) 0.5 kg; Penetration enhancer: borneol 0.5 kg; Purified water q.s. to 200 kg.

[0071] Preparation process: Weigh purified water and add it to a blender. Stir at a speed of 15 rpm. Add glycerol, sodium polyglutamate, PVP1788, main component 2 at room temperature and stir for 30 - 60 minutes until evenly mixed; (2) Stop stirring the blender. Transfer the liquid material from step (1) to a dispensing tank and keep it at 70 °C. Stir at a speed of 20 rpm for 20 - 40 minutes. Turn off the heating, seal the dispensing tank well, and let it stand overnight to obtain the aqueous phase; (3) Add silicone oil, Peregal O-25, and cetyl alcohol to an emulsifier. Set the emulsification temperature at 70 °C. Add borneol before emulsification. Wait for 30 - 60 minutes until all the oil phase is dissolved and reaches 70 °C; (4) Vacuum suck in the aqueous phase, homogenize for 7 minutes, emulsify for 15 minutes, cool the liquid material, and fill it.

[0072] Example 9 Emulsion-type film-forming agent and its preparation Main component 1: silicone oil (molecular weight 1200) 10 kg; Main component 2: STL 1.5 kg or ANB 0.6 kg or PWQ 0.9 kg or ENQ 0.6 kg or paeonol 0.75 kg; Emulsifier: Peregal O-25 7.5 kg, Peregal O-9 7.5 kg, cetyl alcohol 5 kg, Tween-80 1.5 kg; Film-forming material: PVP1788 20 kg, PVP0588 5 kg; Humectant: propylene glycol 20 kg, glycerol 5 kg, sodium polyglutamate (30000 Da) 0.5 kg; Penetration enhancer: menthol 1.0 kg; Purified water q.s. to 200 kg.

[0073] Preparation process: (1) Weigh purified water and add it to a blender. Stir at a speed of 15 rpm. Add glycerol, propylene glycol, polyvinyl alcohol, sodium polyglutamate, and main component 2 at room temperature and stir for 30 - 60 minutes until evenly mixed; (2) Stop the mixer, transfer the liquid material from step (1) to a dispensing tank, keep it warm at 75°C, stir at a speed of 20 rpm for 20 - 40 minutes, turn off the heating, seal the dispensing tank well, and let it stand overnight to obtain the aqueous phase; (3) Add silicone oil, Peregal O, cetyl alcohol, and Tween - 80 to an emulsifier, set the emulsification temperature at 75°C, add menthol before emulsification, and wait for 30 - 60 minutes until all the oil phase is dissolved and reaches 75°C; (4) Vacuum - suck in the aqueous phase, homogenize for 10 minutes, emulsify for 18 minutes, cool the liquid material, and fill it.

[0074] Example 10 Emulsion - type film - forming agent and its preparation Main component 1: 5 kg of silicone oil (molecular weight 1500); Main component 2: 0.6 kg of STL or 0.24 kg of ANB or 0.36 kg of PWQ or 0.24 kg of ENQ; or 0.5 kg of gallic acid, 0.25 kg of gallotannic acid; Emulsifier: 22 kg of Peregal O - 25, 7.5 kg of cetyl alcohol, 2.5 kg of Tween - 80; Film - forming material: 20 kg of PVP0588, 2.5 kg of hydroxypropyl methylcellulose (75000 mPa·s); Humectant: 10 kg of propylene glycol, 15 kg of glycerol, 0.5 kg of sodium polyglutamate; Penetration enhancer: 0.5 kg of menthol; Purified water is added to make 200 kg.

[0075] Preparation process: (1) Weigh 80% of the purified water in the formula amount, add it to a mixer, stir at a speed of 15 rpm, add propylene glycol, glycerol, sodium polyglutamate, PVP0588, and main component 2 at room temperature, and stir for 30 - 60 minutes until well - mixed; Take the remaining purified water, heat it to boiling, add hydroxypropyl methylcellulose with stirring until it is in a suspended state, and cool it to room temperature; (2) Stop the mixer, transfer the liquid material from step (1) to a dispensing tank, keep it warm at 75°C, stir at a speed of 20 rpm for 20 - 40 minutes, turn off the heating, seal the dispensing tank well, and let it stand overnight to obtain the aqueous phase; (3) Add silicone oil, Peregal O - 25, cetyl alcohol, and Tween - 80 to an emulsifier, set the emulsification temperature at 75°C, add menthol before emulsification, and wait for 30 - 60 minutes until all the oil phase is dissolved and reaches 75°C.

[0076] (4) Vacuum - suck in the aqueous phase, homogenize for 5 minutes, emulsify for 12 minutes, cool the liquid material, and fill it.

[0077] The above scar-removing latex-type coating agent of the present invention can be used daily at 1 to 3 g, 1 to 3 times a day.

[0078] The above examples demonstrated the in vitro and in vivo effects and potential mechanisms of the scar reducer of the present invention; the compatibility and preparation method of the scar-removing external agent of the present invention were described. The common excipients and preparation processes of the scar-removing external agent, including the selection of transdermal absorption promoters, emulsification technology and preparation processes, etc., can be found in reference books such as "Handbook of Pharmaceutical Excipients" published by Chemical Industry Press in 2005 and written by Sheski et al., or the excipient manuals of suppliers; based on reference books or textbooks such as "Pharmaceutical Preparation Technology" published by Chemical Industry Press in 2015 and written by Yu Guanghua et al., substitutions or transformations can be made without departing from the basic idea of the present invention. Based on the examples, similar implementation schemes that can be obtained by those skilled in the art without creative efforts, including simple substitutions of external agents, such as changing the emulsion-type coating agent to an ointment, cream, patch, etc., or substitutions of emulsifiers, penetration enhancers, humectants, film-forming materials, etc., should be regarded as within the protection scope of the present invention.

Claims

1. Use of a compound in the preparation of a scar reducing agent, characterized in that, The compound is 4-ethoxycarbonyl-quinolin-2-one.

2. The application according to claim 1, wherein The scar reducer is an external preparation for topical application to the skin.

3. The application according to claim 2, characterized in that The external preparation for topical application to the skin is selected from one of tinctures, ointments, creams, pastes, aerosols, sprays, lotions, liniments, paints, film-forming agents, gels, and patches.

4. A scar-removing external preparation, wherein the scar-removing external preparation is an emulsion film-forming agent, and the scar-removing external preparation contains a film-forming material, a penetration enhancer, an emulsifier, a humectant and silicone oil, and is characterized in that, The external scar-removing agent further contains 4-ethoxycarbonyl-quinolin-2-one.

5. The scar-removing external preparation according to claim 4, characterized in that, The film-forming material is selected from at least one of polyvinyl alcohol, sodium alginate, and hydroxypropyl methylcellulose; the penetration enhancer is selected from at least one of menthol, borneol, wintergreen oil, and azone; the emulsifier is selected from at least one of Peregal, poloxamer, Tween, and cetyl alcohol; the moisturizer is selected from at least one of allantoin, propylene glycol, glycerol, sodium polyglutamate, sodium hyaluronate, and urea; the average molecular weight of the silicone oil is 500 - 6000; the silicone oil is an unmodified silicone oil.

6. The scar-removing external preparation according to claim 4, characterized in that, The weight ratio of the silicone oil to the film-forming material, emulsifier, moisturizer, and penetration enhancer in the external scar-removing agent is: silicone oil 0.5 - 10 parts; film-forming material 10 - 40 parts; emulsifier 1 - 40 parts; moisturizer 5 - 40 parts. Penetration enhancer 0.1 - 2 parts.

7. The scar-removing external preparation according to claim 6, characterized in that, The weight ratio of the silicone oil to 4-ethoxycarbonyl-quinolin-2-one in the external scar-removing agent is: silicone oil 0.5 - 10 parts; 4-ethoxycarbonyl-quinolin-2-one 0.1 - 1.8 parts.

8. The scar-removing external preparation according to claim 7, characterized in that, The weight ratio of the silicone oil to 4-ethoxycarbonyl-quinolin-2-one in the external scar-removing agent is: silicone oil 2 - 10 parts; 4-ethoxycarbonyl-quinolin-2-one 0.12 - 0.36 parts.

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