CN120284962A

CN120284962A - Application of a compound in the preparation of a scar reducing agent and a scar removing external preparation

Info

Publication number: CN120284962A
Application number: CN202510596937.8A
Authority: CN
Inventors: 刘威嘉; 孙浠哲; 杨贵滨
Original assignee: Harbin Ruipai Pharmaceutical Biotechnology Co ltd; Heilongjiang Longde Pharmaceutical Co ltd
Current assignee: Harbin Ruipai Pharmaceutical Biotechnology Co ltd; Heilongjiang Longde Pharmaceutical Co ltd
Priority date: 2023-07-19
Filing date: 2023-07-19
Publication date: 2025-07-11
Also published as: CN120168454A; CN116687945B; CN116687945A

Abstract

The invention belongs to the technical field of skin wound repair, and particularly relates to application of a compound in preparing a scar reducing agent and a scar removing external agent. According to the invention, through analysis of an anti-scar traditional Chinese medicine prescription and research on components and activity, the invention discovers that daucosterol, lithospermum alcohol, claritol and 4-ethoxyformyl-quinolone-2-ketone have the effects of inhibiting proliferation of human keloid fibroblasts and inhibiting TGF-beta 1 induced synthesis of human normal skin fibroblast collagen, have the effect of reducing scars of scar animal models, and can be used as scar reducing agents, in particular to external preparations for local application of skin. The invention further discloses an external preparation for removing scars, namely an emulsion type film coating agent, wherein the main component contains antioxidant and/or any one of daucoside, lithospermum alcohol, salvianolic acid, 4-ethoxyformyl-quinolone-2-ketone besides silicone oil, and the main component has better effect of reducing scars of animal models of scars when the main component is used for local external application of skin.

Description

Application of compound in preparing scar reducing agent and scar removing external agent

Technical Field

The invention belongs to the technical field of skin wound repair, and particularly relates to application of a compound in preparing a scar reducing agent and a scar removing external agent.

Background

Scar formation is a necessary result of wound healing. However, compensatory repaired skin tends to be in a different form than native skin. In the wound repair process, skin fibroblast hyperproliferation and collagen hyperproliferation can easily cause pathological scars, such as hypertrophic scars and keloids, and can influence the beauty and even the skin functions.

Physical means such as surgery and laser have been used for reducing scars, but medicines or cosmetics are more convenient to use, and are the main means for reducing skin scars at present. Jiang Jiang et al, 2008, volume 36, journal of clinical military medical science, 1 st phase of current medical treatment of scar, introduce commonly used scar-reducing agents, also known as scar-reducing agents, scar-removing agents or scar-removing agents, including glucocorticoids, all-trans retinoic acid, pingyangmycin, silicones (silicone oils) and monomeric compounds in traditional Chinese medicine extracts or traditional Chinese medicines. Among them, the scar reducing agents such as glucocorticoid and pingyangmycin which are needed to be injected have the problem of inconvenient use and have great side effects.

In recent years, the potential of traditional Chinese medicines for reducing scars is realized at home and abroad, and research has been advanced to a certain extent. Li Guifeng et al 2014, published in "pharmaceutical treatment progress of pathological scars" at 9 th phase of the book 23 of Chinese cosmetology, reviewed this. The traditional Chinese medicine purple flower burn cream, scald cream, compound scar cream, scar I, scar ointment and the like show good scar reducing effect in clinical application. Onion extracts have scar-reducing effects, and among them, flavones such as quercetin and kaempferol are considered as scar-reducing active ingredients, and onion extracts are widely used in related patents such as CN107496661 a. The Chinese medicinal scar-removing monomer materials which have been studied intensively include tanshinone IIA, asiaticoside, glycyrrhizin, ginsenoside Rg3, oleic acid, linoleic acid, sesamin, curcumin, hyperoside, shikonin, etc. The commercial Meibao scar ointment takes oleic acid, linoleic acid and the like as main components. Shikonin, asiaticoside and the like are widely used in related patents such as CN109620859A and the like. The substances generally play a role in regulating and controlling the proliferation of skin fibroblast and the deposition of collagen through signaling pathways such as TGF-beta, PI3K, retinoic acid receptor and the like, thereby reducing scars. In addition, heat shock protein Hsp90 and the like are also involved in the regulation of skin fibroblast proliferation and collagen synthesis. Tang Xiaoyan et al 2017, volume 48 of new medicine, research progress on the relationship between heat shock proteins and pathological scar formation, et al.

Because of the side effects of western medicines for injection, the problems of inconvenient administration and the like, the traditional Chinese medicine and monomer compound scar removing products have the problems of more components, complex compatibility and the like, and the novel scar reducing agent and the scar removing external agent are still development hot spots in the fields of medicines and cosmetics.

Disclosure of Invention

Aiming at the current situation and the existing problems of scar reducing agents and scar removing external agents, the invention analyzes and researches the prescription rules, components and the like of clinical scar removing traditional Chinese medicine compounds, discovers a plurality of novel scar reducing compounds with regulating and controlling effects on skin fibroblast proliferation and collagen synthesis, and develops the scar removing external agents which are convenient to use, and can be used as medicines or cosmetics for preventing or reducing scars, such as hypertrophic scars or keloids in pathological scars.

Accordingly, the present invention first provides the use of a compound comprising daucosterol, lithospermol, violaxadiol, 4-ethoxyformyl-quinolin-2-one for the preparation of a scar reducing agent. Wherein the daucosterol (Sitogluside) has molecular formula of C ₃₅H₆₀O₆, molecular weight of 576.8g/mol, and CAS of 474-58-8, and is contained in Prunellae Spica, fructus Bruceae, spina Gleditsiae, etc. Shikonin (also called shikonin, shikonin; arnebinol) has a molecular formula of C ₁₆H₂₀O₂, a molecular weight of 244.33g/mol, and CAS of 87064-17-3, and is present in radix Arnebiae etc. traditional Chinese medicine. Salvianic acid A (also called as Salvianic acid A C; przewaquinone C) has molecular formula C ₁₈H₁₆O₄, molecular weight 296.3g/mol, CAS ratio 189290-30-0, and is present in radix Salviae Miltiorrhizae. 4-ethoxyformyl-quinolone-2-one (4-Ethoxycarbonyl-2-quinolone) with molecular formula of C ₁₂H₁₁NO₃, molecular weight of 217.22g/mol, CAS of 5466-27-3, and is contained in fructus Bruceae.

The external and skin local smearing researches show that the compound has the effects of inhibiting proliferation of human keloid fibroblasts and inhibiting collagen synthesis of human normal skin fibroblasts induced by TGF-beta 1, has the effect of inhibiting scar hyperplasia of a rabbit ear hyperplasia scar model, and has the effects of reducing scars and promoting healing to a certain extent for rabbit contusion wounds and incision suture wounds. Thus suitable for topical application to the skin, including topical injection or topical application.

Preferably, the scar reducing agent is an external preparation for topical application to skin, including but not limited to, a tincture, ointment, cream, paste, aerosol, spray, lotion, liniment, paint, film, gel, patch for external use.

The invention further researches that the invention takes the silicone oil and the antioxidant as main components, or takes any one of the daucosterol, the lithospermol, the salvianolic acid and the 4-ethoxyformyl-quinolone-2-ketone as the main component, thereby having better inhibition effect on the scar hyperplasia of the rabbit ear hyperplasia scar model.

The invention further improves the scar-removing external preparation and invents an emulsion type film coating agent. The main components of the scar removing external agent are silicone oil and an antioxidant, or silicone oil is added with any one of daucosterol, lithospermol, salviol and 4-ethoxyformyl-quinolone-2-ketone as the main components, or silicone oil and an antioxidant are added with any one of daucosterol, lithospermol, salviol and 4-ethoxyformyl-quinolone-2-ketone as the main components. The scar-removing external agent further comprises a film forming material, a penetration enhancer, an emulsifying agent and a humectant.

Wherein the film forming material can be selected from at least one of polyvinyl alcohol (including but not limited to PVA0588, PVA1788, PVA 1799), sodium alginate, and hydroxypropyl methylcellulose, the permeation enhancer can be selected from at least one of menthol, borneolum Syntheticum, 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, and 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, and the antioxidant can be selected from at least one of gallic acid, tea polyphenols, gallnut tannic acid, paeonol, vitamin C, and vitamin E.

The preferred antioxidant is one of gallic acid, gallnut tannic acid and paeonol.

The preferable molecular weight of the sodium polyglutamate is 20000-30000 Da.

The preferred average molecular weight of the sodium hyaluronate is 50000-700000 Da.

The polymerization degree of the sodium alginate is preferably 80-750.

The preferable viscosity range of the hydroxypropyl methylcellulose 2% aqueous solution at 20 ℃ is 400-200000 mPa.s.

The preferable silicone oil has an average molecular weight of 500-6000, and is non-modified silicone oil.

The scar removing external agent is prepared from, by weight, 0.5-10 parts of silicone oil, 10-40 parts of film forming material, 1-40 parts of emulsifier, 5-40 parts of humectant and 0.1-2 parts of penetration enhancer, wherein the silicone oil is used as a standard and is used as a typical prescription of the scar removing external agent. The preferred scar-removing external agent comprises, by weight, 2-10 parts of silicone oil, 22.5-32.5 parts of film-forming material, 19-32 parts of emulsifier, 15-30 parts of humectant and 0.5-1 part of penetration enhancer.

The anti-scar external agent is prepared from 0.5-10 parts by weight of silicone oil and 0.5-2 parts by weight of antioxidant by taking silicone oil as a standard. The preferred scar-removing external agent comprises 2-10 parts by weight of silicone oil and 0.5-0.75 part by weight of antioxidant. The scar removing external preparation contains film forming material, penetration enhancer, emulsifier and humectant, and can be prepared from silicone oil and antioxidant as main components, or silicone oil and antioxidant as main components, and optionally one of daucosterol, lithospermum alcohol, claritol, and 4-ethoxyformyl-quinolone-2-ketone. The weight proportion of the silicone oil to the film forming material, the penetration enhancer, the emulsifier and the humectant in the scar removing external agent can be the proportion.

The scar removing external preparation is prepared from 0.5-10 parts of silicone oil, 0.1-4.5 parts of daucosterol, 0.1-1.8 parts of lithospermum alcohol, 0.1-2.7 parts of salvianolic acid and 0.1-1.8 parts of 4-ethoxyformyl-quinolone-2-ketone by weight. The preferred scar removing external preparation comprises, by weight, 2-10 parts of silicone oil, 0.3-0.9 part of daucosterol, 0.12-0.36 part of lithospermum alcohol, 0.18-0.54 part of violaxadiol and 0.12-0.36 part of 4-ethoxyformyl-quinolone-2-one. The scar removing external preparation contains film forming material, penetration enhancer, emulsifier and humectant, and can be prepared by adding one of daucosterol, lithospermol, tanshinol and 4-ethoxyformyl-quinolone-2-ketone as main component, or adding one of daucosterol, lithospermol, tanshinol and 4-ethoxyformyl-quinolone-2-ketone as main component. The weight proportion of the silicone oil to the film forming material, the penetration enhancer, the emulsifier and the humectant in the scar removing external agent can be the proportion.

The scar reducing agent and the scar removing external preparation of the invention are not antibiotics, are preferably used after epithelization of skin wounds, and can reduce the stimulation to skin open wounds.

The invention further provides a preparation method of the scar-removing external preparation, which comprises the following steps:

taking purified water, adding a humectant, a film forming material and main components except silicone oil according to parts by weight, and uniformly stirring to prepare a water phase;

adding silicone oil, an emulsifying agent and a penetration enhancer into an emulsifying machine according to parts by weight, and heating and dissolving to prepare an oil phase;

vacuum sucking the water phase into an emulsifying machine, homogenizing and emulsifying, introducing cold water into the interlayer of the emulsifying machine for cooling, and filling.

Advantageous effects

The daucosterol, lithospermum alcohol, claritol and 4-ethoxyformyl-quinolone-2-ketone have the effects of inhibiting proliferation of human keloid fibroblasts, inhibiting synthesis of human normal skin fibroblast collagen induced by TGF-beta 1, inhibiting scar hyperplasia of a rabbit ear hyperplasia scar model, reducing scars and promoting healing to a certain extent on rabbit contusion wounds and incision suture wounds, and acting on multiple targets to facilitate reduction of formula components.

In the scar-removing emulsion type film coating agent, daucosterol, lithospermum alcohol, claritol and 4-ethoxyformyl-quinolone-2-one have good scar hyperplasia inhibiting effect, silicone oil has a certain scar hyperplasia inhibiting effect and can soften scars, an antioxidant has the effect of reducing scar pigmentation after long-term use, a humectant protects wounds, increases hydration environment on the surfaces of the scars, and a penetration enhancer improves penetration of main components into horny layers to increase accumulation of the main components in scar tissues. The main component and the auxiliary material are matched with each other, which is beneficial to improving the scar improving effect of the film coating agent.

The emulsion type film coating agent can form a uniform, transparent, attractive and flexible film after the scar or the wound is dried, can be peeled off, is not polluted by clothes, and is convenient to use.

Drawings

FIG. 1 is an optical density value at 570nm for each group of cells determined by tetramethyl azoazole salt colorimetry after treatment of human keloid fibroblasts with a test compound;

FIG. 2 is a graph showing the results of the soluble and insoluble collagen content measurements of various groups of cells after treatment of human normal skin fibroblasts with the test compounds;

FIG. 3 is a hematoxylin eosin staining chart of typical pathological sections of groups of rabbit ears of a test object-treated rabbit ear proliferative scar model;

FIG. 4 is a graph showing scar elevation index results for a partially treated group after treatment of a rabbit hypertrophic scar model with a test substance;

FIG. 5 is a graph showing the healing of skin and scarring of rabbit bruise wounds, 2-incision linear suture wounds on days 3 and 7 with ENQ administration;

In the figure, DMSO is dimethyl sulfoxide, MDS is asiaticoside, SKN is shikonin, STL is daucosterol, ANB is shikonin, PWQ is salviol, and ENQ is 4-ethoxyformyl-quinolone-2-ketone.

Detailed Description

The technology of the present invention will be clearly and completely explained below by means of some exemplary embodiments. The silicone oil used in the examples is an unmodified silicone oil.

The preparation method comprises the following steps of (1) dimethyl sulfoxide (DMSO), (2) daucosterol (STL), (3) lithospermum Alcohol (ANB), (4) salvianolic acid (PWQ), (5) 4-ethoxyformyl-quinolone-2-ketone (ENQ), (6) asiaticoside (MDS), (7) Shikonin (SKN), (8) Human Keloid Fibroblasts (HKFs), (9) human normal skin fibroblasts (HDFs), (10) heat shock protein (90: HSP 90), (11) Retinoic Acid Receptor (RAR), (12) TGF-beta receptor 1: tbeta R1) and adopting uniform codes for the following chemical substances, cells and protein/receptors.

Modulation of HKFs proliferation by the Compounds of example 1

1.1 Compounds and vehicles

Test compounds MDS (positive control), STL, ANB, PWQ, ENQ, compound vehicle DMSO. The test compound solutions were prepared on-the-fly.

1.2 Cell culture and cell proliferation assay

HKFs (ATCC CRL-1762) was cultured using Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, penicillin (30U/mL), streptomycin (100. Mu.g/mL). After resuscitating subculture, the 4 th generation cells were used for dosing testing. HKFs test compounds (STL, ANB, PWQ, ENQ concentration gradient: 4. Mu.M, 20. Mu.M, 100. Mu.M; MDS concentration: 100. Mu.M) were added to the CO ₂ incubator and incubated for 96h (37 ℃,5% CO ₂).

After completion of the drug addition culture, HKFs was diluted with fresh Dulbecco's modified Eagle medium, inoculated on a cell culture plate at 1X10 ⁵ cells/well, added with 20. Mu.L (5 mg/mL) of tetramethylazo salt (MTT) at 37℃and 5% CO ₂ for 4 hours, the supernatant was discarded, 200. Mu.L of DMSO was added to dissolve MTT, and the optical density value (OD value) at 570nm was measured to evaluate cell proliferation.

1.3 Modulation of HKFs proliferation by Compounds

After addition of each test compound, the OD at 570nm was determined by MTT colorimetry. As shown in FIG. 1, after MDS, STL, ANB, PWQ, ENQ treatment for 96h, the OD values were lower than DMSO (t-test, p < 0.05). Wherein after 96h of STL 100. Mu. M, ANB. Mu. M, PWQ. Mu.100. Mu. M, ENQ. Mu. M, ENQ. Mu.M treatment, the OD values were lower than those of the MDS 100. Mu.M treatment group (t-test, p < 0.05). STL, ANB, PWQ, ENQ has an inhibitory effect on HKFs proliferation, and at the same concentration, the inhibitory effect is higher than MDS and has a certain concentration correlation.

EXAMPLE 2 modulation of HDFs collagen synthesis by Compounds

2.1 Compounds and vehicles

Test compound SKN (positive control), STL, ANB, PWQ, ENQ, compound vehicle DMSO. The test compound solutions were prepared on-the-fly.

TGF-. Beta.1 was dissolved in 0.1% bovine serum albumin solution (w/v) containing 4mM HCl for use.

1.2 Cell culture and collagen Synthesis assay

HDFs (ATCC PCS-201-012) was cultured using Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, penicillin (30U/mL), streptomycin (300. Mu.g/mL). After resuscitating subculture, the 4 th generation cells were used for dosing testing.

HDFs addition of DMSO (blank), TGF-. Beta.1 (model control, 10 ng/ml), HDFs addition of TGF-. Beta.1 (STL, ANB, PWQ, ENQ concentration gradient: 2. Mu.M, 10. Mu.M, 50. Mu.M; SKN concentration: 2. Mu.M) and test compound. After dosing HDFs was incubated in a CO ₂ incubator for 72h (37 ℃, 5% CO ₂).

After the completion of the drug addition culture, the soluble and insoluble collagens in the culture medium and the cells were measured respectively using the Sircol collagen measurement kit according to the operation instructions.

1.3 Effect of Compounds on HDFs collagen Synthesis

The results of the measurement of soluble and insoluble collagens in the medium and cells after addition of each test compound are shown in FIGS. 2A and 2B, respectively. TGF-. Beta.1 induces HDFs increases in soluble and insoluble collagen content (t-test, p < 0.05). SKN, STL, ANB, PWQ, ENQ can reduce TGF-beta 1 induced increases in soluble and insoluble collagen levels (t-test, p < 0.05). The soluble collagen content of STL concentration groups, ANB concentration groups of 10 mu M and 50 mu M concentration groups, PWQ concentration groups of 10 mu M and 50 mu M concentration groups and ENQ concentration groups is lower than that of SKN2 mu M group (t test, p < 0.05), and the soluble collagen content of ANB2 mu M, PWQ mu M group and SKN2 mu M group is not significantly different (t test, p > 0.05). The insoluble collagen content was lower in STL, ANB, PWQ, ENQ concentration groups than in SKN2 μm group (t-test, p < 0.05). STL, ANB, PWQ, ENQ has an inhibitory effect on the increase of HDFs collagen synthesis induced by TGF-beta 1, and has a certain concentration dependence.

Example 3 protein/receptor docking test of Compounds

This example demonstrates the protein/receptor docking test method and results for a portion of the compounds during the study. The selected proteins/receptors are those involved in regulating fibroblast proliferation and collagen synthesis.

3.1 Method 1

Method 1 is referred to : "Synthesis, docking, and in vitro studies of some substituted bischalcones on acid and alkaline phosphatases"( authors Singh M, raghav N, journal splitting and page numbers MEDICINAL CHEMISTRY RESEARCH 2014, 23:1781-1788.

3.2 Test results and conclusions of method 1

The protein/receptor of method 1 includes HSP90, RAR, T beta R1, and the compound includes STL, ANB, PWQ, ENQ. The main test results are shown in Table 1 (1 bit after the decimal point is retained by the value).

Table 1 test results of method 1

The binding energy (about the sum of Van der Waals force and hydrogen bond energy) of the four compounds and HSP90, RAR and TβR1 is lower than-89, and is similar to or lower than the binding energy of the corresponding protein/receptor of the known HSP90 small molecule inhibitor 17-acrylamide-17-demethoxygeldanamycin and derivatives and RAR small molecule agonist all-trans retinoic acid, and the binding stability is better. Wherein the intermolecular van der Waals forces are inversely related to the molecular weight of the compound. The intermolecular hydrogen bond energy between the protein/receptor and the four compounds is the lowest in ENQ and the highest in 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).

3.4 Supplemental test

3.4.1 Method 2

Method 2 is referred to :"Possible SARS-coronavirus 2 inhibitor revealed by simulated molecular docking to viral main protease and host toll-like receptor"( authors HuX, cai X, etc., journal volumes and pages "Future Virology", 2020, 15 (1): 359-368).

3.4.2 Test results and conclusions of method 2

The proteins/receptors of method 2 include HSP90, RAR, TβR1 (PDB ID is shown in Table 1), and the compounds include STL, ANB, PWQ, ENQ. The binding energy of the four compounds with HSP90, RAR and TβR1 is lower than-5 kcal/mol, and the lowest binding energy is-10.18 kcal/mol, so that the four compounds have good binding effect.

The above results, combined with example 1 and example 2, demonstrate that compound STL, ANB, PWQ, ENQ can exert regulatory skin fibroblast proliferation and collagen synthesis activity by targeting HSP90, RAR, tβr1.

Example 4 animal model investigation of hypertrophic scar of Rabbit ear

This example demonstrates the effect of compounds examined during the course of the study on hypertrophic scarring.

4.1 Laboratory animals and reagents

Male New Zealand white rabbits (2.5 kg-3.0 kg). Feeding after 3 days of pre-examination in a normal class environment.

Control and reagents:

The control and reagents set are shown in Table 2. Wherein, the reagent containing silicone oil, such as Gallic Acid (GA) and the reagent of silicone oil, the silicone oil is singly administered for metering, and GA is singly administered in the presence of DMSO as solvent. The treatment of the other silicone oil-containing reagents was the same. DMSO is used as an auxiliary material control, and GA+silicone oil is used as a positive control.

TABLE 2 control and reagent settings, solvents and dosage forms

The injection 1 comprises that GA is gallic acid, the molecular weight of silicone oil is 1000, the injection 2 comprises that the volume dose of the reagent containing silicone oil is calculated according to the total volume of DMSO and silicone oil, and the injection 3 comprises that the volume dose of the reagent containing silicone oil is 100mg/kg/d, and the table indicates the reagent doses of components except silicone oil.

4.2 Method

4.2.1 Model fabrication

The method is described in the "establishment of a model of the proliferation scar of rabbit ear" (the author has young cattle, niu Yong dare, chen Yanshang; journal volume and page number "Chinese medical engineering", 2004, 12 (5): 4). And (5) establishing a model according to a manufacturing method of the rabbit ear hypertrophic scar model. The ear of New Zealand white rabbits are anesthetized by pentobarbital for dehairing and conventional disinfection, and the ear is punctured on the ventral surface of a single side ear, so that 5 circular wound surfaces with the diameter of 10mm and direct cartilage are manufactured. Wound healing is observed weekly after wound establishment, and the wound is randomly grouped and dosed after the wound is proliferative.

4.2.2 Grouping and administration and observation

The model New Zealand white rabbits are randomly divided into 18 groups, each group comprises 2 wound surfaces, the model is controlled by about itself (the ears on which the wound surfaces are established are the model side and the ears on which the wound surfaces are not established are the normal control side), the ears on each model side are respectively coated with 4.1 of the control and test agents on the wound surfaces according to the doses of the table 2, each animal is coated twice daily (at intervals of 4-5 hours), the continuous treatment is carried out for 3 weeks, and the local recovery condition of scars is observed every week.

Animals were sacrificed after 3 weeks of dosing, wound surface sites and surrounding and normal ear samples were taken, 10% formalin fixed, paraffin embedded to make 5 μm sections, hematoxylin eosin stained, pathological observation was performed, and scar elevation index (Scar elevation index, SEI) =scar surface peak to cartilage surface height difference/scar surrounding normal skin surface to cartilage surface height difference was measured under a 10-fold mirror.

4.3 Results

After 3 weeks of administration, the staining pattern of the pathological section typical of normal ear and model wound ear is shown in fig. 3. The SEI of the auxiliary material control group after molding is obviously higher than that of the normal control ear (t test, p < 0.05). Positive control group, STL medium dose group and high dose group, ANB medium dose group and high dose group, PWQ medium dose group and high dose group, ENQ medium dose group and high dose group, STL+silicone oil group, ANB+silicone oil group, PWQ+silicone oil group, ENQ+silicone oil group SEI are lower than auxiliary material control (t test, p < 0.05), new blood vessel and cell infiltration area in scar are few, cell arrangement rule.

Wherein the STL high dose group, ANB high dose group, PWQ high dose group, ENQ high dose group, stl+ silicone oil group, anb+ silicone oil group, pwq+ silicone oil group, enq+ silicone oil group SEI was lower than the positive control group (t-test, p < 0.05). The stl+silicone oil group, anb+silicone oil group, pwq+silicone oil group, enq+silicone oil group SEI were lower than the STL high dose group, ANB high dose group, PWQ high dose group, ENQ high dose group, respectively, although the difference was not significant (t-test, p > 0.05). The STL low dose group, ANB low dose group, PWQ low dose group, ANB low dose group SEI were lower than the adjuvant control, the difference was not significant (t-test, p > 0.05). Figure 4 shows the scar SEI contrast for each model set with SEI lower than the adjuvant control.

EXAMPLE 5 investigation and safety of healing of Rabbit contusion wounds and incision-suture wounds

This example demonstrates the effect and safety of compounds examined during the study on the healing of contusion wounds and incision-suture wounds.

5.1 Laboratory animals and reagents

Experimental animals Male New Zealand white rabbits (2.5 kg-3.0 kg). Feeding after 3 days of pre-examination in a normal class environment.

The control and test agent comprises 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).

5.2 Method

5.2.1 Model fabrication

The number of rabbits is 2, and the skin depilatory on the back of the left and right sides is dehaired. 1 round contusion (i.e. bruise) wound and 2 incision linear suture wounds with the same area or length are respectively manufactured on the left side and the right side of each rabbit two days after dehairing.

5.2.2 Grouping

The experiment is that the left and right control of the experiment is that the reagent is smeared on the right side and the blank auxiliary material is smeared on the left side. The medicine is applied for 7 days externally, 1 time daily, and 4 layers of disinfection gauze are covered after the medicine is applied to prevent the medicine from being rubbed off.

5.2.3 Observations index

Observing the changes of the treatment and control parts before and after 3-7 days of administration, recording the allergic symptoms of the administration parts during and within 7 days of administration, and adverse reactions of the cardiovascular, respiratory and central nervous systems of animals.

5.3 Results

The skin of the rabbit does not have obvious allergic symptoms in the administration period and within 7 days of administration, and the activity coordination ability of the rabbit does not have abnormality, cardiovascular and respiratory system and other obvious adverse reactions in the administration period and within 7 days of administration.

In the self-control, the healing condition of the contusion wound on the test drug side and the suture wound on the incision is better than that of the blank auxiliary material side, the scar is smaller than that of the blank auxiliary material side, and the wound area or length of the test drug side is smaller than or about equal to that of the blank auxiliary material side after 3 to 7 days of administration under the same condition. Wherein the dmso+enq reagent contusion wound area and incision suture wound length are smaller than the blank adjuvant side (see fig. 5, dark part is wound site). The test reagent does not delay the healing of contusion wounds, incision suture wounds or facilitate accelerating wound healing.

Example 6 emulsion type film coating agent and preparation

2Kg of silicone oil (molecular weight 1000);

main ingredient 2 is STL0.3kg or ANB0.12kg or PWQ0.18kg or ENQ0.12kg or GA0.5kg;

Emulsifying agent, peregal O-25 20kg, cetyl alcohol 5 kg;

PVP0588 30kg and sodium alginate (polymerization degree 600) 2.5kg;

Humectant comprising glycerol 15kg, urea 0.25kg, and sodium polyglutamate (20000 Da) 0.25kg;

0.5kg of menthol as a penetration enhancer;

purified water was added to 200kg.

The preparation process comprises the following steps:

(1) Weighing purified water, adding the purified water into a stirrer, stirring at 15rpm, adding glycerol, urea, sodium polyglutamate, PVP0588, sodium alginate and main component 2 at room temperature, and stirring for 30-60 minutes until uniformly mixing;

(2) Stopping stirring by a stirrer, transferring the feed liquid in the step (1) to a liquid preparation tank for heat preservation at the temperature of 72 ℃, stirring at the stirring speed of 20rpm for 20-40 minutes, closing the heating, sealing the liquid preparation tank, and standing overnight to obtain a water phase;

(3) Adding silicone oil, peregal O-25 and cetyl alcohol into an emulsifying machine, setting the emulsifying temperature to 72 ℃, adding menthol before emulsification, and completely dissolving the oil phase for 30-60 minutes until the oil phase reaches 72 ℃;

(4) Vacuum sucking water phase, homogenizing for 5 min, emulsifying for 16 min, cooling the liquid, and packaging.

Example 7 emulsion type film coating agent and preparation

Main component 1 silicone oil (molecular weight 1800) 5kg;

main ingredient 2 is STL0.6kg or ANB0.24kg or PWQ0.36kg or ENQ0.24kg or gallnut tannic acid 0.75kg;

emulsifying agent, peregal O-9 15kg, tween-80.5 kg, cetyl alcohol 5 kg;

PVP1788 25kg as film forming material;

15kg of propylene glycol and 15kg of glycerol;

0.5kg of borneol as a penetration enhancer;

purified water was added to 200kg.

The preparation process comprises the following steps:

(1) Weighing purified water, adding the purified water into a stirrer, stirring at a speed of 15rpm, adding propylene glycol, glycerol, PVP1788 and main component 2 at room temperature, and stirring for 30-60 minutes until the materials are uniformly mixed;

(2) Stopping stirring by a stirrer, transferring the feed liquid in the step (1) to a liquid preparation tank, preserving heat at 75 ℃, stirring for 20-40 minutes at a stirring speed of 20rpm, closing the heating, sealing the liquid preparation tank, and standing overnight to obtain a water phase;

(3) Adding silicone oil, peregal O-9, cetyl alcohol and tween-80 into an emulsifying machine, setting the emulsifying temperature to 75 ℃, adding borneol before emulsification, and completely dissolving the oil phase for 30-60 minutes until the oil phase reaches 75 ℃;

(4) Vacuum sucking water phase, homogenizing for 8 min, emulsifying for 15 min, cooling the liquid, and packaging.

Example 8 emulsion type film coating agent and preparation

Main component 1 silicone oil (molecular weight 2000) 5kg;

Main ingredient 2 is STL0.6kg or ANB0.24kg or PWQ0.36kg or ENQ0.24kg or tea polyphenols 0.75kg;

Emulsifying agent, namely peregal O-25-10 kg, peregal O-9-5 kg and cetyl alcohol 4kg;

PVP1788 25kg as film forming material;

Humectant, glycerol 25kg, sodium polyglutamate (30000 Da) 0.5kg;

0.5kg of borneol as a penetration enhancer;

Purified water was added to 200kg.

The preparation process comprises the following steps:

Weighing purified water, adding into a stirrer, stirring at 15rpm, adding glycerol, sodium polyglutamate, PVP1788 at room temperature,

The main component 2 is stirred for 30-60 minutes until evenly mixed;

(2) Stopping stirring by a stirrer, transferring the feed liquid in the step (1) to a liquid preparation tank, maintaining the temperature at 70 ℃, stirring at 20rpm for 20-40 minutes, closing the heating, sealing the liquid preparation tank, and standing overnight to obtain a water phase;

(3) Adding silicone oil, peregal O-25 and cetyl alcohol into an emulsifying machine, setting the emulsifying temperature to be 70 ℃, adding borneol before emulsification, and completely dissolving the oil phase for 30-60 minutes until the oil phase reaches 70 ℃;

(4) Vacuum sucking water phase, homogenizing for 7min, emulsifying for 15 min, cooling the liquid, and packaging.

Example 9 emulsion type film coating agent and preparation

10Kg of silicone oil (molecular weight 1200) as main component 1;

main component 2 is STL1.5kg or ANB0.6kg or PWQ0.9kg or ENQ0.6kg or paeonol 0.75kg;

Emulsifying agent, namely peregal O-25.5 kg, peregal O-9.5 kg, cetyl alcohol 5kg and tween-80 1.5kg;

PVP1788 20kg and PVP0588 kg;

humectant comprising propylene glycol 20kg, glycerol 5kg, and sodium polyglutamate (30000 Da) 0.5kg;

1.0kg of menthol as a penetration enhancer;

Purified water was added to 200kg.

The preparation process comprises the following steps:

(1) Weighing purified water, adding the purified water into a stirrer, stirring at 15rpm, adding glycerol, propylene glycol, polyvinyl alcohol, sodium polyglutamate and main component 2 at room temperature, and stirring for 30-60 minutes until the mixture is uniformly mixed;

(3) Adding silicone oil, peregal, cetyl alcohol and tween-80 into an emulsifying machine, setting the emulsifying temperature to 75 ℃, adding menthol before emulsification, and completely dissolving the oil phase for 30-60 minutes until the oil phase reaches 75 ℃;

(4) Vacuum sucking water phase, homogenizing for 10 min, emulsifying for 18 min, cooling the liquid, and packaging.

Example 10 emulsion type film coating agent and preparation

Main component 1 silicone oil (molecular weight 1500) 5kg;

Main component 2 comprises STL0.6kg, ANB0.24kg, PWQ0.36kg, ENQ0.24kg, or gallic acid 0.5 kg,

Gallnut tannic acid 0.25kg;

emulsifying agent, peregal O-25 22kg, cetyl alcohol 7.5 kg, tween-80 2.5kg;

PVP0588 20kg, hydroxypropyl methylcellulose (75000 mPa.s) 2.5kg;

Humectant, namely 10kg of propylene glycol, 15kg of glycerol and 0.5kg of sodium polyglutamate;

0.5kg of menthol as a penetration enhancer;

Purified water was added to 200kg.

The preparation process comprises the following steps:

(1) Weighing purified water with the formula amount of 80%, adding a stirrer, stirring at 15rpm, adding propylene glycol, glycerol, sodium polyglutamate, PVP0588 and main component 2 at room temperature, stirring for 30-60 minutes until the mixture is uniform;

(3) Adding silicone oil, peregal O-25, cetyl alcohol and tween-80 into an emulsifying machine, setting the emulsifying temperature to 75 ℃, adding menthol before emulsification, and completely dissolving the oil phase for 30-60 minutes until the oil phase reaches 75 ℃.

(4) Vacuum sucking water phase, homogenizing for 5min, emulsifying for 12 min, cooling the liquid, and packaging.

The scar-removing emulsion type film coating agent can be used for 1-3 g daily and 1-3 times daily.

The embodiment shows the in-vitro and in-vivo effects and the potential mechanism of the scar reducing agent, and describes compatibility of the scar removing external agent, a preparation method and the like. The common auxiliary materials and the preparation process of the scar removing external agent comprise the selection of a percutaneous absorption promoter, an emulsification technology, a preparation process and the like, and can be replaced or changed by a person skilled in the art on the basis of a tool book or a textbook and the like of a medical auxiliary material handbook and the like published by Sessier et al, chemical industry Press 2005, a tool book or a textbook of a supplier and the like published by Yu Anhua et al, chemical industry press 2015. On the basis of examples, similar embodiments, which are available to the person skilled in the art without inventive effort, including simple replacement of external agents, such as the replacement of emulsion-type film coating agents with ointments, creams, patches, etc., or replacement of emulsifiers, permeation enhancers, moisturizers, film forming materials, etc., should be considered as the scope of the present invention.

Claims

1. Use of a compound for the preparation of a scar reducing agent, wherein the compound is 4-ethoxyformyl-quinolone-2-one.

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

3. The use according to claim 2, wherein the topical preparation for topical application to the skin is selected from one of tincture, ointment, cream, paste, aerosol, spray, lotion, liniment, film, gel, patch.

4. The scar-removing external preparation is an emulsion type film coating agent, and contains a film forming material, a penetration enhancer, an emulsifying agent, a humectant and silicone oil, and is characterized by further containing 4-ethoxyformyl-quinolone-2-ketone.

5. The scar-removing external preparation according to claim 4, wherein the film-forming material is at least one selected from polyvinyl alcohol, sodium alginate and hydroxypropyl methylcellulose, the penetration enhancer is at least one selected from menthol, borneol, wintergreen oil and azone, the emulsifier is at least one selected from peregal, poloxamer, tween and cetyl alcohol, the humectant is at least one selected from allantoin, propylene glycol, glycerol, sodium polyglutamate, sodium hyaluronate and urea, the silicone oil has an average molecular weight of 500-6000, and the silicone oil is non-modified silicone oil.

6. The scar-removing external preparation according to claim 4, wherein the weight ratio of silicone oil to film-forming materials, emulsifiers, moisturizers and penetration enhancers in the scar-removing external preparation is 0.5-10 parts of silicone oil, 10-40 parts of film-forming materials, 1-40 parts of emulsifiers, 5-40 parts of moisturizers and 0.1-2 parts of penetration enhancers.

7. The scar-removing external preparation according to claim 6, wherein the weight ratio of silicone oil to 4-ethoxyformyl-quinolone-2-one in the scar-removing external preparation is 0.5-10 parts of silicone oil and 0.1-1.8 parts of 4-ethoxyformyl-quinolone-2-one.

8. The scar-removing external preparation according to claim 7, wherein the weight ratio of silicone oil to 4-ethoxyformyl-quinolone-2-one in the scar-removing external preparation is 2-10 parts of silicone oil and 0.12-0.36 part of 4-ethoxyformyl-quinolone-2-one.