Composition capable of intervening collagen fibrosis as well as preparation method and application thereof

Abstract

The invention relates to a composition capable of intervening collagen fibrosis and a preparation method and application thereof.The composition comprises gallic acid or gallic acid derivatives, and when the composition is used for repairing damaged skin, fibroblast differentiation can be intervened; the composition can also comprise silicone and Ectoine, and the three components cooperate to interfere with collagen fibrosis, so that the composition can be further used for treating or preventing skin diseases such as scars, scleroderma or skin photoaging caused by collagen fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of topical formulation technology, and in particular relates to a composition capable of intervening in collagen fibrosis, its preparation method, and its application. Background Technology

[0002] Collagen fibers are the main structural proteins of human skin, a major component of the dermis and extracellular matrix (ECM), and the tissue basis of skin pathology. They are mainly divided into type I and type III collagen. Type I collagen accounts for about 80% of collagen composition. In the dermis, it aggregates into large, parallel fiber bundles that interweave to form a network. It is an important component in maintaining skin tension and bearing tensile strength, giving the skin a plump and full appearance. Type III collagen consists of immature, finer collagen fibers and is a major component of the reticular fiber structure. Many diseases are caused by or produce collagen fibrosis, such as scleroderma, scarring, or photoaging.

[0003] Scleroderma is an autoimmune rheumatic disease of unknown cause, characterized by severe multi-system connective tissue fibrosis. The pathogenesis of scleroderma is not fully understood, but it is currently believed that immune system dysfunction activates and secretes various cytokines, producing multiple autoantibodies that cause damage and activation of vascular endothelial cells, stimulating fibroblasts to synthesize excessive collagen, leading to fibrosis of the blood vessel walls and tissues. The activation and transformation of fibroblasts into myofibroblasts is a core element in the pathogenesis of scleroderma. Myofibroblasts secrete large amounts of extracellular matrix components, such as collagen, resulting in fibrosis of the skin and other organs. TGF-β (transforming growth factor-β) is a key factor in the fibrotic process of scleroderma. It exacerbates the fibrotic response by promoting fibroblast proliferation, transformation, and collagen secretion. Patients with scleroderma often experience damage to small blood vessels. Damage to endothelial cells promotes fibrosis and proliferation of the vascular endothelium, reduces vascular plasticity, and leads to narrowing and occlusion of small blood vessels. After damage, endothelial cells can transform into interstitial cells (such as myofibroblasts), which is one of the important mechanisms of fibrosis.

[0004] Excessive proliferation of fibroblasts and excessive collagen deposition during scar formation lead to pathological scars. Hypertrophic scars are more common in wounds that extend into the dermis. Compared to normal scars, hypertrophic scars exhibit more active fibroblast proliferation and more pronounced collagen deposition, resulting in more significant dermal fibrosis. Collagen fibers are produced throughout the wound healing process and are a key factor in scar formation. Under physiological conditions, fibroblasts and their differentiated myofibroblasts, which possess contractile properties, gradually die off during the initial stages of wound healing. Under pathological conditions, myofibroblast function mutates, producing and depositing large amounts of collagen fibers. The continued presence and abnormal function of myofibroblasts ultimately lead to the formation of hypertrophic scars.

[0005] Photoaging of the skin is primarily caused by prolonged exposure to ultraviolet (UV) radiation. UV radiation activates multiple signaling pathways in the skin, inhibiting collagen synthesis in fibroblasts. Normal collagen fibers are neatly and orderly arranged, providing solid support for the skin. However, during photoaging, UV radiation alters the structure of collagen fibers, making them disordered, loose, and even broken. Simultaneously, abnormal collagen fibers accumulate, forming amorphous substances that replace the normal collagen fiber network—a manifestation of collagen fibrosis. This fibrotic collagen not only loses its normal physiological function but also affects the skin's appearance, causing wrinkles, sagging, and other signs of aging.

[0006] Current drug treatments for collagen fibrosis mainly focus on inhibiting inflammatory responses and regulating cytokines, but existing methods have shortcomings in terms of efficacy, safety, and applicability. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a composition capable of intervening in collagen fibrosis, its preparation method, and its application.

[0008] The technical solution adopted in this invention is: a composition capable of intervening in collagen fibrosis, comprising gallic acid or gallic acid derivatives, silicone and ectoine.

[0009] Preferably, the mixture comprises, by mass parts, 0.01-10 gallic acid or gallic acid derivatives, 0.01-90 silicone, and 0.01-5 ectoine.

[0010] Preferably, the gallic acid derivative is propyl gallate or bismuth subgallate.

[0011] Preferably, the silicone is polydimethylsiloxane, cyclopentadimethylsiloxane, or dimethyl silicone oil.

[0012] An agent capable of intervening in collagen fibrillation, comprising a composition capable of intervening in collagen fibrillation.

[0013] Preferably, the formulation is a cream or a hydrocolloid; the cream includes emulsions and ointments.

[0014] Preferably, when the formulation is an ointment, the composition comprises, by weight parts, 0.01-10 parts gallic acid or gallic acid derivatives and 20-70 parts silicone; or, the composition comprises 0.01-10 parts gallic acid or gallic acid derivatives, 20-70 parts silicone and 0.01-5 parts ectoine.

[0015] When the formulation is a hydrocolloid, the composition comprises, by mass parts, 0.01-10 parts gallic acid or gallic acid derivatives and 0.01-10 parts silicone; or, the composition comprises 0.01-10 parts gallic acid or gallic acid derivatives, 0.01-10 parts silicone and 0.01-5 parts ectoine.

[0016] Preferably, when the formulation is a cream, it further includes one or more of excipients, emollients, thickeners, pH adjusters, preservatives, and water; when the formulation is an emulsion, it further includes an emulsifier; and when the formulation is an ointment, it does not include an emulsifier.

[0017] Alternatively, when the formulation is a hydrocolloid, it may also include one or more of the following: water-absorbing materials, adhesive substances, and plasticizers.

[0018] Preferably, the excipient is petrolatum or cocoa butter; the emollient includes any one or a combination of at least two of glycerin, propylene glycol, and trehalose; the emulsifier includes any one or a combination of at least two of lactic acid fatty acid glycerides, ES-5300, and glyceryl stearate; the pH adjuster includes triethanolamine, sodium hydroxide, lactic acid, and sodium bicarbonate; the preservative includes methylparaben and a combination of phenoxyethanol; the thickener in the emulsifier may be one or a combination of carbomer, sodium carboxymethyl cellulose, and xanthan gum; the thickener in the ointment may be one or a combination of sodium carboxymethyl cellulose, sodium alginate, and hydroxyethyl cellulose.

[0019] Preferably, the absorbent material is one or more of sodium carboxymethyl cellulose, gelatin, pectin, sodium alginate, hydroxyethyl cellulose, hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, and sodium hydroxypropyl methyl cellulose; the adhesive substance includes one or two of thermoplastic rubber and hot melt adhesive; and the plasticizer is one or more of dioctyl adipate, dioctyl terephthalate, and liquid paraffin.

[0020] The use of compositions or preparations capable of intervening in collagen fibrosis in the preparation of products for treating collagen fibrosis diseases.

[0021] Preferably, it is applicable to skin diseases involving collagen fibrosis, specifically scars, scleroderma, or photoaging of the skin.

[0022] The advantages and positive effects of this invention are: it provides a composition that can be used to intervene in collagen fiber formation, which effectively controls the differentiation and growth of collagen fibers during skin growth through the synergistic effect of gallic acid or gallic acid derivatives with silicone and ectoine; based on this feature, formulations including this composition can be used in drugs related to scars, scleroderma or photoaging of the skin. Attached Figure Description

[0023] Figure 1 HE staining images of different compositions on the pathological observation of skin lesions in hypertrophic scar mice;

[0024] Figure 2 Masson staining images of different compositions and their formulations on the pathological observation of skin lesions in hypertrophic scar mice;

[0025] Figure 3 Relative mRNA levels of COL1A1, COL1A2, α-SMA, and TGF-β in hypertrophic scar mouse skin of different compositions and their formulations; Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001;

[0026] Figure 4 HE staining images of different compositions and their formulations on the pathological observation of skin lesions in scleroderma mice;

[0027] Figure 5 Masson staining images of different compositions and their formulations on the pathological observation of skin lesions in scleroderma mice;

[0028] Figure 6 The mRNA expression of COL1A1, COL1A2, TGF-β, and α-SMA in scleroderma using different compositions and their formulations; Note: Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001; Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0029] Figure 7 The activities of different compositions and their formulations on MDA, CAT and SOD in the serum of photoaged mice; Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001;

[0030] Figure 8 Relative mRNA levels of COL1A1, COL1A2, α-SMA, and TGF-β in photoaged mouse skin of different compositions and their formulations; Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001. Detailed Implementation

[0031] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0032] This invention relates to a composition capable of intervening in collagen fibrosis, its preparation method, and its application. The composition includes gallic acid or gallic acid derivatives, as well as silicone. The two components work synergistically to intervene in collagen fibrosis and can be further used for the treatment or prevention of skin diseases caused by collagen fibrosis, such as scars, scleroderma, or photoaging of the skin.

[0033] Gallic acid derivatives are propyl gallate or bismuth subgallate, and silicone is polydimethylsiloxane, cyclopentasiloxane, or dimethyl silicone oil; the mass ratio of gallic acid or gallic acid derivatives to silicone is related to the product dosage form. When the dosage form is a cream, the composition includes 0.01-10 parts of gallic acid or gallic acid derivatives and 20-90 parts of silicone by mass; specifically, it may include 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 parts of gallic acid or gallic acid derivatives and 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 parts of silicone; when the dosage form is a hydrocolloid, the mass ratio is... The composition comprises, by weight, 0.01-10 parts gallic acid or gallic acid derivatives and 0.01-10 parts silicone; specifically, it may comprise 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 parts gallic acid or gallic acid derivatives and 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 parts silicone. When propyl gallate is added to the composition, a better intervention effect on collagen fibrosis can be achieved. In some embodiments of the present invention, the composition also includes ectoine; through the synergistic effect of ectoine with gallic acid or gallic acid derivatives and silicone, the intervention effect on collagen fibrosis can be further enhanced. The mass ratio of gallic acid or gallic acid derivatives, silicone, and ectoine is related to the product dosage form.When the dosage form is a cream, the composition comprises, by weight, 0.01-10 parts gallic acid or gallic acid derivatives, 20-90 parts silicone, and 0.01-5 parts ectoine; specifically, it may include 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 parts gallic acid or gallic acid derivatives, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 parts silicone, and 0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 parts ectoine; when the dosage form is a hydrocolloid, according to... The composition comprises, by weight parts, 0.01-10 parts gallic acid or gallic acid derivatives, 0.01-10 parts silicone, and 0.01-5 parts ectoine; specifically, it may comprise 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 parts gallic acid or gallic acid derivatives, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 parts silicone, and 0.01, 0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 parts ectoine.

[0034] Gallic acid or its derivatives possess signal inhibition, cell regulation, and anti-inflammatory and antioxidant effects. Gallic acid or its derivatives can target and block the TGF-β / Smad signaling pathway (a core driver of fibrosis), downregulate the transcription of COL1A1 and COL1A2 (key genes for type I collagen synthesis), and reduce excessive collagen synthesis. They can also inhibit fibroblast proliferation and differentiation into myofibroblasts (high expression of α-SMA, mediating tissue contracture). Furthermore, they can scavenge reactive oxygen species (ROS), inhibit NF-κB pathway activation, and reduce the release of pro-inflammatory / pro-fibrotic factors such as IL-1β and TNF-α. Silicone can form a breathable protective film on the skin surface, maintaining a local moist microenvironment, reducing moisture loss and external stimuli. It can also regulate the rate of collagen fiber synthesis, inhibit disordered cross-linking and accumulation of collagen, and promote the alignment of collagen bundles in a direction parallel to the normal skin surface. Simultaneously, it reduces the contractile activity of myofibroblasts, alleviating scar contractures or skin hardening. Ectoin stabilizes cell membrane structure and repairs DNA damage (such as UV-induced photoaging damage and vascular endothelial damage in scleroderma) through its "molecular chaperone" function; it downregulates profibrotic factors such as PDGF and TGF-β, inhibits the abnormal increase of MMPs (matrix metalloproteinases, which lead to collagen degradation in photoaging); it rebuilds the skin barrier, restores the function of keratinocytes / vascular endothelial cells, and enhances the skin's own ability to resist damage.

[0035] Gallic acid or its derivatives work in conjunction with silicone and ectoine to intervene in fibroblasts from different angles, regulating the generation of fibroblasts in terms of quantity and interfering with their regular arrangement to form a normal skin tissue structure. The three active ingredients complement each other and work together in the process of skin repair and skin growth to solve a variety of skin problems. They have a prominent regulatory effect in skin repair or prevention of skin diseases, forming an effective intervention.

[0036] To better leverage the intervention effects of gallic acid or gallic acid derivatives, silicone, and ectoine on collagen fibers in the composition, the composition can be mixed with other excipients to form an emulsion or hydrocolloid for easy application. Emulsions can be further classified into emulsions or ointments. When the formulation is an emulsion, it may also include one or more excipients, emollients, thickeners, pH adjusters, preservatives, and water. When the formulation also includes an emulsifier, it forms an emulsion formulation; when the formulation does not include an emulsifier, it forms an ointment formulation. The emollients include any one or a combination of at least two of glycerin, propylene glycol, and trehalose; the emulsifiers include any one or a combination of at least two of silicone oil emulsifiers, lactic acid fatty acid glycerides, ES-5300, and glyceryl stearate; the pH adjusters include triethanolamine, sodium hydroxide, lactic acid, and sodium bicarbonate; the preservatives include methylparaben and phenoxyethanol, or a combination of two; the excipients are petrolatum or cocoa butter; the thickeners in the emulsions may be one or a combination of carbomer, sodium carboxymethyl cellulose, and xanthan gum; and the thickeners in the ointments may be one or a combination of sodium carboxymethyl cellulose, sodium alginate, and hydroxyethyl cellulose.

[0037] When the formulation is a hydrocolloid, the composition also includes an absorbent material and / or a viscous substance, as well as a plasticizer. The absorbent material is one or more of sodium carboxymethyl cellulose, gelatin, pectin, sodium alginate, hydroxyethyl cellulose, hydroxypropyl cellulose, croscarmellose sodium carboxymethyl cellulose, and sodium hydroxypropyl methyl cellulose; the viscous substance includes one or two of thermoplastic rubber and hot melt adhesive; the plasticizer is one or more of dioctyl adipate, dioctyl terephthalate, and liquid paraffin.

[0038] Experiments have shown that formulations containing gallic acid or gallic acid derivatives, silicone, and ectoine, when used in mouse models of scarring, scleroderma, or photoaging, exhibit significant improvements in dermal thickness, fibroblast proliferation activity, and collagen deposition. These formulations can reduce scar extent and inhibit the formation of hypertrophic scars; they can suppress excessive fibroblast growth, thereby improving the progression of scleroderma; and during photoaging, they control collagen fiber formation, reducing abnormal collagen fiber accumulation and decreasing the degree of photoaging.

[0039] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in the drawings are performed according to the corresponding product instructions. Unless otherwise specified, all instruments, reagents, and consumables used in the examples can be purchased from commercial companies. All experimental data are quantitative data and were processed using SPSS 20.0 statistical software. All data were found to follow a normal distribution and are expressed as mean ± standard deviation (x ± s). For comparisons of means from multiple groups, one-way ANOVA was used to determine homogeneity of variance, and the LSD method was used for pairwise comparisons. P < 0.05 was considered statistically significant.

[0040] Example 1: Ointment containing gallic acid or gallic acid derivatives

[0041] Bismuth subgallate and petrolatum were mixed in a mass ratio of 0.3:6 and heated and stirred at a temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Then, the mixture was gradually cooled to 30℃ while stirring to obtain the experimental group 1-1 formulation.

[0042] Gallic acid and petrolatum were mixed in a mass ratio of 0.3:6 and heated and stirred at a temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Then, the mixture was gradually cooled to 30℃ while stirring to obtain preparations 1-2 of the experimental group.

[0043] Propyl gallate and petrolatum were mixed at a mass ratio of 0.3:6 and heated and stirred at a temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Then, the mixture was gradually cooled to 30℃ while stirring to obtain preparations 1-3 of the experimental group.

[0044] Example 2: Silicone-containing ointment

[0045] Polydimethylsiloxane and petrolatum were mixed at a mass ratio of 1:20 and heated and stirred at a temperature of 40℃-45℃ and a stirring speed of 30-45r / min for 15-20min to obtain preparation 2 of experimental group.

[0046] Example 3: Ointment containing ectoine

[0047] Hot melt adhesive and plasticizer were added at a mass ratio of 11:1, heated to 100-150℃, and stirred until uniformly mixed. Then, water-absorbing granules and ectoine were added at a mass ratio of 8:0.25, and stirred until uniformly mixed to obtain preparation 3 of experimental group.

[0048] Example 4: Ointment containing silicone and ectoine

[0049] Polydimethylsiloxane and petrolatum were mixed and heated at a mass ratio of 1:20, with a heating temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Ectoin (polydimethylsiloxane, petrolatum and ectoin in a mass ratio of 1:20:0.25) was added, mixed and stirred evenly, and then cooled to 30℃ to obtain preparation 4 of experimental group.

[0050] Example 5: Ointment containing gallic acid and silicone

[0051] Polydimethylsiloxane and petrolatum were mixed and heated at a mass ratio of 1:20, with a heating temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Gallic acid (polydimethylsiloxane, petrolatum and gallic acid in a mass ratio of 1:20:0.3) was added, mixed and stirred evenly, and then cooled to 30℃ to obtain preparation 5 of experimental group.

[0052] Example 6: Ointment containing gallic acid or gallic acid derivatives, silicone, and ectoine

[0053] Polydimethylsiloxane and petrolatum were mixed and heated at a mass ratio of 1:20, with a heating temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Bismuth hypogalactate and ectoine were added (the mass ratio of polydimethylsiloxane, petrolatum, bismuth hypogalactate and ectoine was 1:20:0.3:0.25), mixed and stirred evenly, and then cooled to 30℃ to obtain the experimental group 6-1 formulation.

[0054] Polydimethylsiloxane and petrolatum were mixed and heated at a mass ratio of 1:20, with a heating temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Gallic acid and ectoine (polydimethylsiloxane, petrolatum, gallic acid and ectoine in a mass ratio of 1:20:0.3:0.25) were added, mixed and stirred evenly, and then cooled to 30℃ to obtain the experimental group 6-2 formulation.

[0055] Polydimethylsiloxane and petrolatum were mixed and heated at a mass ratio of 1:20, with a heating temperature of 40℃-45℃ and a stirring speed of 30-45 r / min for 15-20 min. Propyl gallate and ectoine were added (the mass ratio of polydimethylsiloxane, petrolatum, propyl gallate and ectoine was 1:20:0.3:0.25), mixed and stirred evenly, and then cooled to 30℃ to obtain the experimental group 6-3 formulation.

[0056] Example 7: Hydrocolloid containing propyl gallate, silicone and ectoine

[0057] Hot melt adhesive, dioctyl adipate, and polydimethylsiloxane were added in a mass ratio of 11:1:0.5. The mixture was heated to 100-150℃ and stirred until homogeneous. Then, gelatin, propyl gallate, and ectoine were added in a mass ratio of 7:1:0.5 and stirred until homogeneous to obtain the experimental group 7-1 formulation.

[0058] Hot melt adhesive, dioctyl adipate, and polydimethylsiloxane were added in a mass ratio of 11:1:0.7. The mixture was heated to 100-150℃ and stirred until it was evenly mixed. Then, gelatin, propyl gallate, and ectoine were added in a mass ratio of 7:1:0.3 and stirred until it was evenly mixed to obtain the experimental group 7-2 preparation.

[0059] Hot melt adhesive, dioctyl adipate, and polydimethylsiloxane were added in a mass ratio of 11:1:0.3. The mixture was heated to 100-150℃ and stirred until homogeneous. Then, gelatin, propyl gallate, and ectoine were added in a mass ratio of 7:1:0.7 and stirred until homogeneous to obtain the experimental group 7-3 formulation.

[0060] Example 8: Ointments containing different proportions of propyl gallate, silicone, and ectoine

[0061] To study the effect of different ratios of active ingredients on the efficacy of the formulation, ointments with different ratios were prepared sequentially using propyl gallate, silicone, and ectoin as raw materials.

[0062] The ointment was prepared according to the method of Example 6. Formulation 8-1 was prepared when the mass ratio of propyl gallate, silicone, and ectoine was 0.01:90:5; formulation 8-2 was prepared when the mass ratio was 5:50:3; formulation 8-3 was prepared when the mass ratio was 10:20:0.01; formulation 8-4 was prepared when the mass ratio was 3:60:1; and formulation 8-5 was prepared when the mass ratio was 0.1:70:4.

[0063] Example 9: Hydrocolloids containing different proportions of propyl gallate, silicone, and ectoine

[0064] Hydrocolloids were prepared according to the method of Example 7. When the mass ratio of propyl gallate, silicone, and ectoine was 0.01:10:3, the formulation of Experimental Example 9-1 was prepared; when the mass ratio of propyl gallate, silicone, and ectoine was 7:8:0.01, the formulation of Experimental Example 9-2 was prepared; when the mass ratio of propyl gallate, silicone, and ectoine was 10:0.01:5, the formulation of Experimental Example 9-3 was prepared; and when the mass ratio of propyl gallate, silicone, and ectoine was 0.1:2.5:0.1, the formulation of Experimental Example 9-4 was prepared.

[0065] Example 10: Emulsion containing propyl gallate, silicone, and ectoine

[0066] Preparation of phase A: Polydimethylsiloxane and silicone oil emulsifier are mixed and heated at a mass ratio of 10:1, with stirring at 70℃-85℃ and a stirring speed of 30-45 r / min for 15-20 min. Preparation of phase B: Glycerin, water, propyl gallate, and ectoine are mixed and stirred at a mass ratio of 1:10:0.2:0.1 until dissolved. Phases A and B are mixed at a mass ratio of 3.5:6.5, homogenized, and stirred for 5 min at a stirring speed of 40-45 r / min for 40-50 min. The mixture is then cooled to obtain the final phase.

[0067] The efficacy of the formulations prepared in Examples 1-10 was verified. When comparing the efficacy of the formulations prepared in Examples 1-8, the mass fraction of silicone was the same in each formulation; when silicone was not present in the composition, the mass fraction of gallic acid or its derivative was the same; when silicone and gallic acid were both absent in the formulation, the mass fraction of ectoine was the same.

[0068] Example 11: Application of a composition formulation capable of intervening in collagen fibrosis in scar repair

[0069] 11.1 Establishment of a mouse model of hypertrophic scarring

[0070] Healthy female SPF-grade C57 mice aged 6-8 weeks were used. After acclimatization, the back hair of the mice was removed using an electric shaver and depilatory cream. The mice were observed after shaving. One day after hair removal, the following procedures were performed: After the mice were stretched and fasted, the posterior midline (a), the midaxillary line (b), and the line (c) between the two lines were marked. The upper back muscles on line (c) were then sutured to the ribs and intercostal muscles below line (b) using 6-0 nylon sutures. Finally, a 1 cm linear incision was made along the posterior midline (a) between the fixed skin. The mice were observed for two weeks postoperatively.

[0071] 11.2 Application of the composition formulation

[0072] Two weeks later, the modeling mice were randomly divided into groups and treated with different drugs according to the group. The preparations prepared in Examples 1-7 were applied to the scars of the modeling mice. A control group was set up and treated with Bacon silicone gel. There was also a model group that did not receive any treatment. The experimental group and the control group were treated with drugs 3 times a day, with each group receiving 30 μl per dose for 14 days.

[0073] Four weeks after modeling, on day 29, mice in each group were euthanized by cervical dislocation. Skin tissue from the lesions on the back of the mice was cut off with sterile scissors, and the tissue was quickly collected and marked. A portion of the tissue was placed in 4% tissue fixative for preservation, and the other portion was placed in cryovials and flash-frozen in liquid nitrogen for 1 hour before being transferred to a -80°C water tank for cryopreservation for subsequent experiments.

[0074] 11.3 HE staining and dermal thickness detection of skin lesions on the back of mice

[0075] Skin samples were taken from the target lesion on the back of mice, cut into 0.2 × 0.4 cm pieces, and stained with hematoxylin and eosin (HE) using a kit. The epidermal thickness and tissue inflammation of the pathological tissue were observed under a light microscope. The HE staining results are shown below. Figure 1 As shown, the scar tissue in the model group mice was significantly higher than the surrounding skin, with epidermal hyperplasia, no reticular ridges, active and significantly thickened dermis, and abundant fibrous connective tissue hyperplasia beneath the epidermis. The number of skin appendages such as hair follicles was reduced, or even absent, accompanied by inflammatory cell infiltration and microvascular proliferation. Compared with the model group, the scar tissue in each experimental group and the control group was reduced to varying degrees. The six preparations in experimental groups 6 and 7 showed significantly better scar reduction effects than those in experimental groups 1-5, demonstrating that preparations containing gallic acid or gallic acid derivatives, silicone, and ectoine can better inhibit scar formation at damaged skin sites. Among these six preparations, experimental group 7-3 showed the best scar reduction effect, with significantly reduced epidermal and dermal hyperplasia, reduced connective tissue, more intact skin appendages, and reduced inflammatory cell infiltration. Experimental groups 7-2 and 7-1 showed better scar reduction effects than those in experimental group 6, with significantly reduced epidermal-dermal hyperplasia and reduced connective tissue. Comparing the effects of each formulation in experimental groups 1-5, experimental group 5, which added gallic acid and silicone, and experimental group 4, which added silicone and ectoine, showed better effects than experimental groups 1-3. Among them, the effect of experimental group 2 was comparable to that of the commercially available products in the control group. All groups in experimental group 1 were also effective in reducing scars, but the effect was slightly less than that of experimental group 2. The ectoine ointment in experimental group 3 had the worst effect.

[0076] HE-stained sections were observed under a fluorescence microscope. Five fields of view were randomly selected, and the dermal thickness of the mouse dorsal skin was measured using a micrometer attached to the microscope. The mean value of the five fields of view was taken as the final measurement result. The results are shown in Table 1. After modeling, the dermal thickness of the model group mice was significantly increased. Compared with the model group, the dermal thickness of the experimental groups and the control group mice was significantly reduced (P<0.05, P<0.01, P<0.001, P<0.0001).

[0077] Table 1. Effects of different compositions and their formulations on the dermal thickness of skin lesions on the back of mice with hypertrophic scars.

[0078]

[0079] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the control group, ## P<0.01, # P<0.05.

[0080] Besides the overall effect shown by HE staining, analysis of the above data revealed that, in terms of dermal thickness at the lesion site, experimental group 4 showed a better effect than the combined effect of experimental groups 2 and 3, demonstrating a synergistic effect between polydimethylsiloxane and ectoine. Experimental group 5 showed a better effect than the combined effect of experimental groups 1-3 and experimental group 2, demonstrating a synergistic effect between polydimethylsiloxane and propyl gallate. Experiments in experimental groups 4 and 5 demonstrated that the combination of propyl gallate and silicone was more effective than the combination of silicone and ectoine. Data from experimental groups 6 and 7 showed that the combination of gallic acid or gallic acid derivatives, silicone, and ectoine was more effective than the sum of the effects of the three active ingredients alone, and greater than the sum of the effects of a combination of two active ingredients or a single active ingredient. For example, the effects of experimental groups 6-3 were greater than those of experimental groups 1-3 and experimental group 2. The sum of the effects of experimental group 3 was greater than the sum of the effects of experimental groups 1-3 and group 4, and greater than the sum of the effects of experimental groups 5 and group 3. The reduction effect of experimental group 2 was not significantly different from that of the control group, indicating that it had a significant effect on improving the microstructure of skin tissue morphology. The reduction effects of experimental groups 1 and 3 were slightly less than those of the control group.

[0081] 11.4 Masson staining detection of skin lesions on the back of mice

[0082] Skin samples were taken from the target lesion on the back of mice, cut into 0.2 × 0.4 cm pieces, and stained with Masson's stain using a kit. Collagen deposition and arrangement in each group were observed under an optical microscope. The Masson staining results are shown below. Figure 2As shown, the model group mice exhibited collagen hyperplasia, with magnification revealing a large number of fibroblasts, collagen fibers of varying thicknesses, and disordered yet densely distributed collagen bundles. Compared to the model group, both the experimental and control groups showed varying degrees of reduction in collagen fiber count.

[0083] 11.5 Detection of fibroblast count in dorsal skin lesions of mice

[0084] When fibroblast proliferation becomes uncontrolled, it synthesizes and secretes large amounts of extracellular matrix, leading to abnormal thickening of scar tissue. The proliferation was assessed by detecting the number of fibroblasts in the skin lesions on the back of mice. On day 29 after treatment, skin tissue specimens from the backs of mice in each group were selected to prepare tissue sections. These sections were observed using a regular optical microscope. Five complete, non-repeating high-power fields were randomly selected from each section to count the number of fibroblasts in the granulation tissue, and the mean value was used as the measurement for that section.

[0085] The results are shown in Table 2. The model group mice showed excessive growth of fibroblasts. Compared with the model group, the experimental group and the control group mice showed a significant reduction in fibroblasts (P<0.001, P<0.0001), thereby inhibiting scar formation.

[0086] Table 2. Effects of different compositions and their formulations on the number of fibroblasts in the dorsal skin lesions of mice with hypertrophic scars.

[0087]

[0088] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the control group, ## P<0.01, # P<0.05.

[0089] Analysis of the above data revealed that the effects of each experimental group's formulation on the number of fibroblasts in the affected skin were consistent with the trend in dermal thickness. The effect of experimental group 4 was superior to the combined effects of experimental groups 2 and 3, demonstrating a synergistic effect between polydimethylsiloxane and ectoine. The effect of experimental group 5 was superior to the combined effects of experimental groups 1-3 and experimental group 2, demonstrating a synergistic effect between polydimethylsiloxane and propyl gallate. The comparison between experimental groups 4 and 5 demonstrated that the combination of propyl gallate and silicone was more effective than the combination of silicone and ectoine. The effects of experimental groups 6 and 7 were significantly superior to the other groups, demonstrating that the combination of gallic acid or gallic acid derivatives, silicone, and ectoine was more effective than the sum of the effects of the three active ingredients alone, and greater than the sum of the effects of a combination of two active ingredients or the sum of the effects of one active ingredient.

[0090] 11.6 Quantitative Real-Time PCR Detection

[0091] In scars, TGF-β is a core signal for initiating and maintaining fibrosis, and is the most important pro-fibrotic cytokine, considered the "master switch" for scar formation. α-SMA is a marker of functionally activated myofibroblasts, responsible for scar contractures; its expression enables cells to have contractile functions similar to smooth muscle cells, and is a major cause of pathological scar contractures, hardening, and functional impairment. Overexpression of COL1A1 and COL1A2 is the molecular basis for excessive type I collagen deposition in scar tissue, directly leading to the mechanical strength and non-compliance of scars. These four factors together constitute the key molecular axis of scar formation and are the focus of current basic research and clinical treatment development for scars. This study analyzed the mRNA expression of COL1A1, COL1A2, α-SMA, and TGF-β in hypertrophic scar mouse wounds using different compositions and their formulations.

[0092] Take an appropriate amount of skin and place it in a 2ml RNase-free EP tube. Add 500μl of Trizol reagent and an appropriate amount of grinding beads to each tube, and homogenize rapidly in a tissue homogenizer. After standing until the foam disappears, centrifuge at 12000r for 15min at 4℃. Transfer the supernatant to a new RNase-free EP tube, add 100μl of chloroform, vortex rapidly until homogeneous, and let stand for 10min. Centrifuge at 12000r for 15min at 4℃. Slowly aspirate the upper aqueous phase from 200μl of the EP tube to a new RNase-free EP tube, add 200μl of isopropanol, mix by inverting, let stand for 10min, and centrifuge at 12000r for 10min at 4℃. Pour out the liquid, invert and air dry, add 200μl of 75% anhydrous ethanol to the tube, vortex until homogeneous, centrifuge at 12000r for 5min at 4℃, discard the supernatant, and allow the precipitate to dry at room temperature in a laminar flow hood. Dissolve the precipitate in 20μl of DEPC water.

[0093] RNA was reverse transcribed into cDNA using a reverse transcription kit. GAPDH was used as an internal control, SYBR Green was used as the dye, and the cDNA was used as a template for quantitative real-time PCR amplification. The reaction volume was 20 μL, and the amplification conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 40 amplification cycles; melting curve amplification: 95℃ for 5 s, 60℃ for 5 s; extension at 95℃ for 5 s. The results were calculated using 2... －ΔΔCt The relative expression levels were analyzed to compare the differences in the expression of COL1A1, COL1A2, α-SMA, and TGF-β among the groups.

[0094] The results are as follows Figure 3As shown, the model group exhibited abnormally high expression of COL1A1, COL1A2, α-SMA, and TGF-β mRNA, indicating that tissue injury releases large amounts of TGF-β, stimulating high activation of fibroblasts, leading to high expression of α-SMA, excessive contractile force, and scar contracture. High expression of COL1A1 and COL1A2 resulted in excessive deposition of type I collagen, causing hardening and thickening of scar tissue. Compared with the model group, the levels of the aforementioned mRNAs in the experimental groups and the control group were significantly higher. On average, there were varying degrees of significant reduction (P<0.05, P<0.01, P<0.001, P<0.0001); compared with the control group, experimental groups 2, 4, 5, 6, and 7 significantly inhibited the expression of COL1A1, COL1A2, α-SMA, and TGF-β (P<0.05, P<0.01, P<0.001), with experimental groups 7-3 showing the best effect. This indicates that it can both inhibit excessive collagen synthesis and reduce deposition, and restore normal contraction, thereby reducing scar formation.

[0095] In scar suppression, gallic acid or its derivatives can sever the "signal source" of fibrosis, directly targeting the TGF-β / Smad pathway, inhibiting the core driving signals of fibroblast activation, reducing the mRNA expression of COL1A1 and COL1A2, and decreasing type I collagen synthesis at the molecular level. Simultaneously, gallic acid or its derivatives can alleviate the continuous stimulation of fibroblasts by local inflammation by inhibiting inflammatory factors such as IL-1β and TNF-α, preventing excessive fibroblast proliferation. Silicone can form a protective film on the scar surface, maintaining a moist local environment and preventing scar hyperplasia caused by dryness. Silicone can also inhibit the contractile function of myofibroblasts, reduce the functional activity of α-SMA, and decrease scar contracture. Furthermore, silicone can guide collagen fibers to align parallel to the skin surface, preventing disordered accumulation that leads to scar hardening and protrusion. Ectoin can repair damaged keratinocytes at the site of injury in the "trauma microenvironment", restore the skin barrier function, and reduce the interference of external stimuli on scar repair; at the same time, it downregulates profibrotic factors such as PDGF (platelet-derived growth factor) and assists gallic acid in inhibiting fibroblast proliferation, avoiding the limitations of a single component that only blocks signals and cannot improve the microenvironment.

[0096] Through systematic research and testing on the condition of scarred skin, significant differences were found in the efficacy of different compositions and their formulations in improving scar condition. Based on a comprehensive evaluation of various test indicators, the efficacy ranking of the experimental groups was as follows: Experimental Group 7 > Experimental Group 6 > Experimental Group 5 > Experimental Group 4 > Experimental Group 2 > Control Group > Experimental Group 1 > Experimental Group 3 > Model Group.

[0097] Among them, experimental group 7 showed significantly better results than experimental groups 4 and 5 in improving skin fibrosis, inhibiting collagen deposition, and regulating fibroblast activation, indicating that the combination of the three active substances produced a significant synergistic effect, which was better than any combination of two. Gallic acid derivatives and silicone alone, and ectoine alone, have some effect on hypertrophic scars, but their effects are far less than any combination of two, and far less than the effect of the combination of the three active substances. The three active substances cover the entire process of "inflammatory activation → fibroblast activation → collagen deposition → scar contracture," blocking signaling through gallic acid or its derivatives, optimizing collagen structure through silicone, and repairing the microenvironment through ectoine. This overcomes the shortcomings of single components (such as silicone alone being unable to block TGF-β-driven collagen synthesis, and gallic acid alone lacking regulation of collagen arrangement).

[0098] Through the synergistic effect of gallic acid derivatives, silicone, and ectoine, the process of collagen fibrosis can be significantly inhibited, showing excellent efficacy in the treatment of hypertrophic scars. Among them, experimental group 6 (basic ratio of gallic acid derivative + polydimethylsiloxane + ectoine ointment) and experimental groups 7-3 (hydrocolloid formulation with the same ingredients) showed the best results, with the hydrocolloid formulation having greater advantages in scar treatment. This composition has flexible formulation and high safety, providing a new and efficient treatment option for collagen fibrosis diseases.

[0099] Example 12: Application of a composition formulation capable of intervening in collagen fibrosis in scleroderma

[0100] 12.1 Establishment of a mouse model of scleroderma and administration of the compound formulation

[0101] Prepare a 100mM potassium dihydrogen phosphate solution with pH 6.2, mix thoroughly, and store at 4°C. Subsequently, prepare an HOCl solution before each injection using a sodium hypochlorite solution:potassium dihydrogen phosphate solution ratio of 1:50.

[0102] Healthy female SPF-grade C57 mice aged 6-8 weeks were randomly divided into groups using a lottery method. After one week of acclimatization, the hair in the central area of ​​the mice's backs, covering an area of ​​approximately 2.0 cm × 2.0 cm, was shaved. Modeling was initiated after confirming the absence of allergic or inflammatory reactions on the back skin. Modeling method: Control group mice were subcutaneously injected with 0.1 mL PBS, while other groups were subcutaneously injected with 0.1 mL of 200 μg / mL bleomycin hydrochloride, once daily for 6 weeks. During modeling, the back skin of the model group mice was not treated in any way. The backs of the other treatment groups were treated with the preparations obtained in Examples 1-7, respectively, while the control group was treated with Centella asiatica ointment. The dosage for each group was 30 μl per administration.

[0103] Six weeks after modeling, changes in the appearance of the mouse skin were observed. In the model group, the injection area on the back of the mice was significantly hardened and adhered, making it difficult to lift, and no new hair growth was observed. In the experimental groups and the control group, the skin on the back of the mice was soft and easy to lift, with slight thickening of the injection area, no hardening or adhesion, and rapid hair growth. Among them, the skin on the back of the mice in experimental groups 6 and 7 was softer, especially the mice in experimental groups 7-3, whose back hair gradually grew and whose skin was the softest and easiest to lift. In experimental groups 1-5, the skin of the mice in experimental groups 5 and 4 was softer than that of the control group. The effect of experimental group 2 was comparable to that of the commercially available product in the control group. All groups in experimental group 1 also showed an effect on alleviating scleroderma, but the effect was slightly less than that of experimental group 2. The ectoine ointment alone in experimental group 3 had the worst effect.

[0104] Six weeks after modeling, mice in each group were euthanized by cervical dislocation. Skin tissue from the lesions on the back of the mice was cut off with sterile scissors, and the tissue was quickly collected and labeled. A portion of the tissue was placed in 4% tissue fixative for preservation, while the other portion was placed in cryovials, flash-frozen in liquid nitrogen for 1 hour, and then transferred to a -80°C water bath for cryopreservation for subsequent experiments.

[0105] 12.2 HE staining and Masson staining of mouse skin tissue

[0106] Mouse skin tissue was stained with hematoxylin and eosin (HE) using a kit. Paraffin sections of skin tissue from each group were dewaxed and hydrated with xylene and alcohol, stained with hematoxylin, differentiated with 1% ethanol, counterstained with 0.5% eosin solution, dehydrated, cleared, and mounted. The HE staining results for each group of mouse skin are shown below. Figure 4 As shown. Mouse skin tissue was stained with Masson stain using a kit. Paraffin sections of skin tissue from each group were dewaxed and hydrated with xylene and alcohol, stained and rinsed with nuclear staining and plasma staining solutions, counterstained with blue counterstaining solution, rinsed thoroughly with anhydrous ethanol, and air-dried. Pathological changes in the skin tissue were observed under an optical microscope (×200). The Masson staining results of mouse skin in each group are shown below. Figure 5 As shown.

[0107] HE and Masson staining results in mice showed that the dermis of the model group mice was significantly thickened, with a large number of lymphocytes and plasma cells infiltrating at the junction of the dermis and subcutaneous tissue. The fat layer was thinned or disappeared, and skin appendages were destroyed, atrophied, and reduced. Collagen was significantly thickened, enlarged, increased in number, and densely arranged. Subdermal blood vessels were enlarged, with significantly thickened walls, narrowed lumens, and even complete blockage. Compared with the model group, the dermis of mice in the experimental groups and the control group was thinner to varying degrees, with a small amount of inflammatory cell infiltration and a decrease in the number of collagen fibers. Among them, experimental groups 6 and 7 were more effective than experimental groups 1-5. The alleviation effect of experimental group 7 on scleroderma was better than that of experimental group 6, with a significant reduction in epidermal and dermal hyperplasia and connective tissue. In particular, experimental groups 7-3 showed the best alleviation effect on scleroderma, with a significant reduction in epidermal and dermal hyperplasia, a reduction in connective tissue, more intact skin appendages, and a reduction in inflammatory cell infiltration. Among experimental groups 1-5, experimental groups 4 and 5 showed better results, significantly better than the control group; the effect of experimental group 2 was comparable to that of commercially available products in the control group; all groups in experimental group 1 were also effective in alleviating scleroderma, but the effect was slightly less than that of experimental group 2; and the ectoine ointment alone in experimental group 3 had the worst effect.

[0108] 12.3 Detection of dermal thickness and fibroblast count in mouse skin

[0109] Masson-stained sections were observed under an Olympus microscope. Dermal thickness was measured using ImagePro Plus software. Six different regions were randomly selected from each photograph for measurement, and the average value was calculated. The results are shown in Table 3.

[0110] Table 3 Comparison of dermal thickness in different groups of mice (x-±s, μm)

[0111]

[0112] Note: Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001; Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

[0113] After successful modeling, skin tissue specimens from each group were cut and observed using a regular optical microscope. Five complete and non-repeating high-power fields of view were randomly selected from each section to count the number of fibroblasts in the skin tissue, and the mean value was taken as the measurement value for that section. The results are shown in Table 4.

[0114] Table 4 Comparison of fibroblast numbers in each group of mice

[0115]

[0116] Note: Compared with the control group, # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001; Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

[0117] Analysis of the data in Tables 3 and 4 revealed similar trends in the effects of each formulation on dermal thickness and the number of fibroblasts in mouse skin. The effect of experimental group 4 was superior to the combined effects of experimental groups 2 and 3, demonstrating a synergistic effect between polydimethylsiloxane and ectoine. The effect of experimental group 5 was superior to the combined effects of experimental groups 1-3 and experimental group 2, demonstrating a synergistic effect between polydimethylsiloxane and propyl gallate. Data from experimental groups 4 and 5 showed that the combination of propyl gallate and silicone was more effective than the combination of silicone and ectoine. The effects of experimental groups 6 and 7 were significantly superior to the other formulations, demonstrating that the combination of gallic acid or gallic acid derivatives, silicone, and ectoine was more effective than the sum of the effects of the three active ingredients alone, and greater than the sum of the effects of a combination of two active ingredients or the sum of the effects of one active ingredient. For example, the effects of experimental groups 6-3 were greater than those of experimental groups 1-3 and experimental group 2. The sum of the effects of experimental group 3 was greater than the sum of the effects of experimental groups 1-3 and group 4, and greater than the sum of the effects of experimental groups 5 and group 3. The reduction effect of experimental group 2 was not significantly different from that of the control group, indicating that it had a significant effect on improving the microstructure of skin tissue morphology. The reduction effects of experimental groups 1 and 3 were slightly less than those of the control group.

[0118] 12.4 Quantitative Real-Time PCR Detection

[0119] In scleroderma, COL1A1, COL1A2, α-SMA, and TGF-β collectively constitute a positive feedback, uncontrolled fibrotic axis under an autoimmune background. TGF-β is the core driver of fibrosis in scleroderma, forming a fibrotic cycle that cannot be stopped spontaneously. α-SMA is a direct marker of the abundant and persistently activated myofibroblasts, and is consistently highly expressed in the dermis and fibrotic organs of scleroderma patients, being a major cause of tissue contracture and hardening. COL1A1 and COL1A2 show significantly and persistently upregulated expression levels in scleroderma fibroblasts, providing the molecular basis for skin and visceral thickening and hardening. Type I collagen is the most abundant collagen type deposited in scleroderma; its abnormally large deposition replaces normal tissue structure, leading to loss of tissue elasticity and impaired function. This study analyzed the effects of different compositions and their formulations on the mRNA expression of COL1A1, COL1A2, TGF-β, and α-SMA in scleroderma.

[0120] Take an appropriate amount of skin and place it in a 2ml RNase-free EP tube. Add 500μl of Trizol reagent and an appropriate amount of grinding beads to each tube, and homogenize rapidly in a tissue homogenizer. After standing until the foam disappears, centrifuge at 12000r for 15min at 4℃. Transfer the supernatant to a new RNase-free EP tube, add 100μl of chloroform, shake rapidly to mix well, and let stand for 10min. Centrifuge at 12000r for 15min at 4℃. Slowly and repeatedly aspirate the upper aqueous phase from the EP tube to a new RNase-free EP tube, add 200μl of isopropanol, mix by inverting, let stand for 10min, and centrifuge at 12000r for 10min at 4℃. Pour out the liquid, invert and air dry, add 200μl of 75% anhydrous ethanol to the tube, shake well, centrifuge at 12000r for 5min at 4℃, discard the supernatant, and allow the precipitate to dry at room temperature in a laminar flow hood. Dissolve the precipitate in 20μl of DEPC water.

[0121] RNA was reverse transcribed into cDNA using a reverse transcription kit. GAPDH was used as an internal control, SYBR Green was used as the dye, and the cDNA was used as a template for quantitative real-time PCR amplification. The reaction volume was 20 μL, and the amplification conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 40 amplification cycles; melting curve amplification: 95℃ for 5 s, 60℃ for 5 s; extension at 95℃ for 5 s. Relative expression levels were analyzed using the 2-ΔΔCt method to compare the differences in gene expression among the groups (collagen mRNA expression levels (COL1A1, COL1A2), TGF-β, and the key myofibroblast marker α-SMA).

[0122] The results are as follows Figure 6As shown, the model group exhibited abnormally high expression of COL1A1, COL1A2, α-SMA, and TGF-β mRNA, indicating that after autoimmune damage, TGF-β was overactivated, fibroblasts were continuously activated, and α-SMA was highly expressed, leading to tissue contracture and hardening, resulting in skin hardening and joint damage. The high expression of COL1A1 and COL1A2 caused abnormal and excessive deposition of type I collagen, replacing normal tissue structure and leading to loss of tissue elasticity and impaired function. Compared with the model group, the mRNA levels of COL1A1 and COL1A2 in experimental groups 4, 5, 6, and 7 were significantly reduced to varying degrees (P<0.05, P<0.01, P<0.001). In experimental groups 6 and 7... The mRNA levels of α-SMA and TGF-β were significantly reduced to varying degrees (P<0.05, P<0.01, P<0.001). Compared with the control group, experimental groups 6 and 7 significantly inhibited the expression of COL1A1, COL1A2, α-SMA and TGF-β (P<0.05, P<0.01, P<0.001), with experimental groups 7-3 showing the best effect. This indicates that they can significantly inhibit the continuous activation of fibroblasts and abnormal collagen synthesis, thereby slowing down the progression of scleroderma.

[0123] The causes of scleroderma include the following pathways: autoimmune abnormalities (T cell activation) lead to the release of large amounts of pro-fibrotic / pro-fibrotic factors (TGF-β, IL-4, IL-13), which further lead to vascular endothelial cell damage (increased permeability, transformation into interstitial cells), continuous activation of fibroblasts and differentiation into myofibroblasts, resulting in a large amount of type I collagen deposition, thickening and hardening of the dermis, accompanied by vascular stenosis or occlusion.

[0124] In the prevention of scleroderma, gallic acid or its derivatives can inhibit "immune-driven fibrosis" by suppressing T cell activation and the release of pro-inflammatory factors such as IL-6 and TNF-α, thereby reducing autoimmune-mediated inflammatory responses. Simultaneously, it potently blocks the TGF-β / Smad pathway, directly inhibiting fibroblast differentiation into myofibroblasts and reducing the overexpression of COL1A1 and COL1A2, thus breaking the chain of "immune activation → fibrosis" at its source. Silicone can protect the "vascular and collagen balance," improving the local skin microenvironment and reducing oxidative stress damage to vascular endothelial cells. It also regulates the collagen deposition rate, preventing excessive cross-linking of collagen fibers in the dermis (a typical feature of scleroderma) and reducing the degree of skin hardening. Furthermore, silicone can inhibit the transformation of vascular endothelial cells into mesenchymal cells (EMT), alleviating vascular stenosis and improving skin blood supply. Ectoin can repair "vascular endothelial damage" and specifically repair damaged vascular endothelial cells in scleroderma. By stabilizing the cell membrane and repairing DNA damage, it restores the barrier function of endothelial cells and reduces interstitial edema caused by increased vascular permeability. At the same time, it downregulates profibrotic factors such as PDGF and assists gallic acid in inhibiting abnormal proliferation of fibroblasts, blocking the positive feedback loop of "vascular damage → fibroblast activation → collagen deposition".

[0125] Through systematic research and testing of the skin condition of scleroderma patients, significant differences were found in the efficacy of different compositions and their formulations in improving the condition of scleroderma. Based on comprehensive testing indicators, the efficacy ranking of the experimental groups was: Experimental Group 7 > Experimental Group 6 > Experimental Group 5 > Experimental Group 4 > Experimental Group 2 > Control Group > Experimental Group 1 > Experimental Group 3 > Model Group. Among these, composition group 7 was significantly superior to experimental groups 4 and 5 in improving skin fibrosis, inhibiting collagen deposition, and regulating fibroblast activation, indicating that the combination of the three active substances produced a significant synergistic effect, which was better than any combination of two. Gallic acid derivatives and silicone alone, and ectoine alone, had some effect on scleroderma, but their effects were far less than any combination of two, and far less than the effect of the combination of the three active substances.

[0126] Targeting the triple pathogenic mechanism of scleroderma, the combination of gallic acid or its derivatives, silicone, and ectoine can significantly delay the progression of scleroderma. Gallic acid primarily targets "immune inflammation and TGF-β signaling," silicone primarily targets "collagen regulation and vascular protection," and ectoine primarily targets "endothelial cell repair." The three work synergistically to block multiple pathways, resulting in a good preventive effect against scleroderma. In the experiment, the dermal thickness of the experimental group 7-3 (365.7±5.128μm) was significantly lower than that of the model group (392.8±5.111μm), and the mRNA expression levels of COL1A1 and TGF-β were significantly reduced, demonstrating that it can effectively delay the progression of scleroderma.

[0127] The composition, through the synergistic effect of gallic acid derivatives, silicone, and ectoine, can significantly inhibit the process of collagen fibrosis and has shown excellent efficacy in the treatment of scleroderma. Experimental group 6 (a basic formula of gallic acid derivatives + polydimethylsiloxane + ectoine ointment) and experimental groups 7-3 (a hydrocolloid formulation with the same ingredients) showed the best results, with the hydrocolloid formulation demonstrating greater advantages in the treatment of scleroderma. This composition offers flexible formulation and high safety, providing a novel and highly effective treatment option for collagen fibrosis.

[0128] Determination of 12,5-hydroxyproline content

[0129] Hydroxyproline is one of the main and key components of collagen tissue and is a unique amino acid in collagen. Its content detection is of great significance for assessing collagen metabolism and related diseases. Since the content of hydroxyproline in collagen is significantly higher than in other proteins, this experiment uses the detection of hydroxyproline content in mouse skin tissue homogenate to reflect the catabolism and metabolism of collagen in vivo. The formulations prepared in Examples 8 and 9 were used as the test drugs and applied to a mouse model of scleroderma, specifically following the experimental procedure in 12.1. The dosage of the formulation was the same in each group in experimental group 8 and the same in each group in experimental group 9.

[0130] After 30 days of drug administration, skin samples were collected from the affected side of all animals in the experimental groups at the pre-defined endpoint of the experimental dissection. The hydroxyproline content of each group's samples was determined using a tissue hydroxyproline assay kit and an enzyme-linked immunosorbent assay (ELISA) reader.

[0131] Based on statistical analysis of various pharmacodynamic indicators, the effects of the formulations in experimental groups 8 and 9 were further evaluated. Statistical analysis methods were selected according to the data distribution characteristics: if the data conformed to a normal distribution, a paired-samples t-test was used to calculate the p-value; if the data did not conform to a normal distribution, a two-tailed Wilcoxon paired t-test was used; when comparing multiple groups of data, analysis of variance (ANOVA) or the Kruskal-Wallis test was used. The statistical significance criterion for the experiment was set at p < 0.05. Specific data on the expression levels of hydroxyproline in the skin of mice in each group are shown in Table 5.

[0132] Table 5

[0133]

[0134] Table 5 shows that after bleomycin modeling, the hydroxyproline content in the skin of mice in the model group increased significantly. Compared with the bleomycin model group, the hydroxyproline content in the skin of mice in the Centella asiatica ointment control group and the nine experimental formulation groups was significantly reduced. This result indicates that formulations containing propyl gallate, silicone, and ectoine can effectively downregulate the expression level of hydroxyproline, thereby helping to alleviate the degree of skin collagen fibrosis.

[0135] Example 13: Application of a composition formulation capable of intervening in collagen fibrosis in skin photoaging.

[0136] 13.1 Establishment of a mouse model of skin photoaging and application of the composition formulation

[0137] Healthy female SPF clean-grade C57 mice aged 6-8 weeks were randomly divided into groups corresponding to different experimental preparations in Examples 1-7, including a control group and a model group.

[0138] After the adaptation feeding period, the back hair of the mice was removed using an electric shaver and depilatory cream. The shaved area was approximately 3cm × 2cm. The mice were observed after shaving. One day after hair removal, the following procedure was performed: The UV therapy device was placed in a cardboard box at a certain height, and the UVB irradiation agent UV-297 probe was used to measure the irradiation count. The irradiation count was recorded after it stabilized. The UV therapy device needed to be preheated for 5 minutes before irradiating the mice. After the light source stabilized, the experimental mice were placed in the irradiation box. Specifically, all groups were given 1 MED (90 mj / cm²) in weeks 1 and 2. 2 UVB dose increased to 2 MED (180 mJ / cm²) in weeks 3 and 4. 2 From week 5 to week 7, the concentration was 2.5 MED (225 mJ / cm). 2 The patients were irradiated at the recommended dose, three times a week, with one irradiation every other day. Drug administration was administered 30 minutes after each UVB irradiation session, and the model was maintained for seven consecutive weeks. The experimental groups received the different formulations described in Examples 1-7, the model groups received no treatment, and the control group received Vitamin E. The dosage for each group was 30 μl per administration.

[0139] Seven weeks after modeling and drug administration, on day 50, blood was collected from the eyes of mice and placed in EP tubes. Then, mice in each group were euthanized by cervical dislocation. Skin tissue from the lesions on the back of the mice was cut off with sterile scissors, collected quickly, and marked. A portion of the tissue was placed in 4% tissue fixative for preservation, and the other portion was placed in cryovials and flash-frozen in liquid nitrogen for 1 hour before being transferred to a -80°C water bath for cryopreservation for subsequent experiments.

[0140] 13.2 Detection of epidermal thickness and fibroblast count in dorsal skin lesions of model mice

[0141] The effects of different compositions and formulations on the epidermal thickness of the dorsal skin of photoaged mice were investigated. Skin sections were prepared from the back of the mice, and the epidermal thickness of the dorsal skin was measured using a micrometer attached to a microscope in five randomly selected fields of view. The average value of the five fields of view was taken as the final result. The results are shown in Table 6.

[0142] Table 6. Effects of different compositions and formulations on epidermal thickness of dorsal skin in photoaged mice.

[0143]

[0144] Note: Compared with the model group, **P<0.01, ***P<0.001, ****P<0.0001; compared with the control group, ## P<0.01, ### P<0.001.

[0145] Fibroblasts are the main cells of the dermis. They maintain the stability of skin structure by regulating collagen synthesis and degradation. However, excessive growth of fibroblasts can lead to tissue overgrowth. This study compared the effects of different compositions and formulations on the number of fibroblasts in dorsal skin lesions of photoaged mice.

[0146] After treatment, on day 50 of the experiment, skin tissue specimens from the backs of mice in each group were selected to prepare tissue sections. These sections were observed using a regular optical microscope. Five complete, non-repeating high-power fields were randomly selected from each section to count the number of fibroblasts in the granulation tissue of the wound, and the mean value was taken as the measurement value for that section. The results are shown in Table 7.

[0147] Table 7. Effects of different compositions and formulations on the number of fibroblasts in dorsal skin lesions of photoaged mice.

[0148]

[0149] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; compared with the control group, # P<0.05, ## P<0.01.

[0150] Analyzing the data in Tables 5 and 6, regarding the effects on epidermal thickness and fibroblast count in the dorsal skin of photoaged mice, the effect of experimental group 4 was superior to the combined effect of experimental groups 2 and 3, demonstrating a synergistic effect between polydimethylsiloxane and ectoine. The effect of experimental group 5 was superior to the combined effect of experimental groups 1-2 and experimental group 2, demonstrating a synergistic effect between polydimethylsiloxane and propyl gallate. The data from experimental groups 4 and 5 demonstrate that the combination of propyl gallate and silicone is more effective than the combination of silicone and ectoine. The effects of experimental groups 6 and 7 were significantly superior to the other groups, demonstrating that the combination of gallic acid or gallic acid derivatives, silicone, and ectoine is more effective than the sum of the effects of the three active ingredients alone, and greater than the sum of the effects of a combination of two active ingredients or the sum of the effects of one active ingredient. For example, the effects of experimental groups 6-3 were greater than those of experimental groups 1-3 and 2; the sum of the effects of experimental group 3 was greater than the sum of the effects of experimental groups 1-3 and 4, and greater than the sum of the effects of experimental groups 5 and 3. The reduction effect of experimental group 5 was not significantly different from that of the control group, indicating that it had a significant effect on improving the microstructure of skin tissue morphology. The reduction effects of experimental groups 1, 2, 3, and 4 were slightly less than those of the control group.

[0151] 13.3 Activities of MDA (malondialdehyde), CAT (catalase), and SOD (superoxide dismutase) in the serum of model mice

[0152] In photoaged mice, changes in serum MDA, CAT, and SOD activities are key indicators for assessing systemic oxidative stress levels. Ultraviolet radiation can directly penetrate the skin, leading to the excessive generation of free radicals from SOD, resulting in its overconsumption. This weakens the body's ability to scavenge core free radicals, creating vulnerabilities in the antioxidant defense system. CAT activity decreases, and hydrogen peroxide accumulates, further exacerbating oxidative damage. These free radicals not only attack skin cells but also enter the bloodstream, triggering systemic lipid peroxidation. Elevated serum MDA levels directly demonstrate that photoaging is not merely a localized skin problem but also a process accompanied by systemic oxidative damage. This study compares the activities of different compositions and formulations of MDA, CAT, and SOD in the serum of photoaged mice.

[0153] After blood was collected from the mouse eye, it was left at room temperature for 30 min and then centrifuged at 4 ℃ and 3500 rpm for 15 min.

[0154] The supernatant serum after centrifugation was used to detect the levels of MDA, CAT, and SOD in mouse serum using an ELISA kit.

[0155] The results are as follows Figure 7As shown, after ultraviolet irradiation, the serum MDA content in the model group increased significantly, indicating severe oxidative damage to the body. The serum CAT and SOD levels decreased, and the antioxidant enzyme activity decreased, indicating that the body's ability to scavenge core free radicals was weakened, further aggravating oxidative damage. Compared with the model group, the serum MDA content in each experimental group and the control group decreased to varying degrees (P<0.05, P<0.01, P<0.001, P<0.0001), and the serum CAT and SOD levels also increased significantly. Compared with the control group, experimental groups 5, 6, and 7 significantly reduced MDA content (P<0.05, P<0.01, P<0.001) and increased the antioxidant enzyme activity of CAT and SOD. Among them, experimental group 7-2 showed the best effect, indicating that it can significantly increase antioxidant levels and reduce ultraviolet-induced lipid peroxidation, thereby alleviating oxidative damage caused by photoaging.

[0156] 13.4 Quantitative real-time PCR detection of skin lesions on the back of model mice

[0157] COL1A1, COL1A2, α-SMA, and TGF-β are core molecules regulating the repair and damage response of the dermal layer of the skin. UV damage to keratinocytes and endothelial cells triggers an inflammatory response, immediately releasing stored TGF-β and stimulating fibroblast activation. The expression level of α-SMA is positively correlated with the degree of fibrosis. Increased synthesis of COL1A1 and COL1A2 leads to abnormal collagen fiber accumulation, forming amorphous substances that replace the normal collagen fiber network. This is accompanied by a large production of MMPs, causing collagen fibers to become sparse, broken, and disordered, resulting in loss of skin strength and elasticity, and the formation of wrinkles. This study compares the effects of different compositions and formulations on the mRNA expression of COL1A1, COL1A2, α-SMA, and TGF-β in photoaged mice.

[0158] Take an appropriate amount of skin and place it in a 2ml RNase-free EP tube. Add 500μl of Trizol reagent and an appropriate amount of grinding beads to each tube, and homogenize rapidly in a tissue homogenizer. After standing until the foam disappears, centrifuge at 12000r for 15min at 4℃. Transfer the supernatant to a new RNase-free EP tube, add 100μl of chloroform, vortex rapidly until homogeneous, and let stand for 10min. Centrifuge at 12000r for 15min at 4℃. Slowly aspirate the upper aqueous phase from 200μl of the EP tube to a new RNase-free EP tube, add 200μl of isopropanol, mix by inverting, let stand for 10min, and centrifuge at 12000r for 10min at 4℃. Pour out the liquid, invert and air dry, add 200μl of 75% anhydrous ethanol to the tube, vortex until homogeneous, centrifuge at 12000r for 5min at 4℃, discard the supernatant, and allow the precipitate to dry at room temperature in a laminar flow hood. Dissolve the precipitate in 20μl of DEPC water.

[0159] RNA was reverse transcribed into cDNA using a reverse transcription kit. GAPDH was used as an internal control, SYBR Green was used as the dye, and the cDNA was used as a template for quantitative real-time PCR amplification. The reaction volume was 20 μL, and the amplification conditions were: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing for 1 min, 40 amplification cycles; melting curve amplification: 95℃ for 5 s, 60℃ for 5 s; extension at 95℃ for 5 s. The results were calculated using 2... －ΔCt Expression levels were analyzed using the ×100% method to compare the differences in the expression of COL1A1, COL1A2, α-SMA, and TGF-β among the groups.

[0160] The results are as follows Figure 8 As shown, after UV irradiation, the mRNAs of COL1A1, COL1A2, α-SMA, and TGF-β in the model group all exhibited abnormally high expression, indicating that after cell damage, inflammatory mediators were released, TGF-β levels increased, fibroblasts were activated, α-SMA was expressed, and wrinkles appeared on the skin. The high expression of COL1A1 and COL1A2 represented increased synthesis of type I collagen and abnormal accumulation of collagen fibers. Compared with the model group, the levels of the above mRNAs in each experimental group and the control group were significantly reduced to varying degrees (P<0.05, P<0.01, P<0.001, P<0.0001). Compared with the control group, experimental groups 5, 6, and 7 significantly inhibited the expression of COL1A1, COL1A2, α-SMA, and TGF-β (P<0.05, P<0.01, P<0.001), with experimental group 7-2 showing the best effect, indicating that it can significantly inhibit excessive collagen synthesis and proliferation, thereby alleviating the photo-aged skin condition.

[0161] Photoaging of the skin is a degenerative process of "UV damage - collagen disorder". Long-term UV exposure can cause skin cells to produce a large amount of ROS. ROS activates the NF-κB pathway and releases inflammatory factors such as IL-1 and IL-6, which leads to fibroblast dysfunction (reduced collagen synthesis but abnormal accumulation, and increased MMPs leading to normal collagen degradation). The collagen fiber structure is damaged, resulting in breakage or disordered arrangement, which manifests as skin laxity, wrinkle formation and decreased elasticity.

[0162] In the prevention of photoaging, gallic acid or its derivatives exhibit potent antioxidant activity against ROS and collagen degradation, scavenging UV-induced ROS, inhibiting NF-κB pathway activation, and reducing the release of inflammatory factors. Simultaneously, it inhibits the activity and expression of MMPs (such as MMP-1 and MMP-3), reducing the degradation and damage of normal collagen fibers. Furthermore, it promotes the synthesis of functional collagen (rather than abnormally accumulated collagen) by fibroblasts, improving the structural integrity of collagen fibers. Silicone can construct a UV protection and collagen alignment barrier, forming a physical barrier on the skin surface that reflects some UV rays, reducing secondary UV damage to skin cells. It also regulates the collagen metabolism balance of fibroblasts, promoting the orderly alignment of collagen fibers parallel to the skin surface, reducing disordered accumulation that leads to rough and sagging skin. Additionally, it can lock in skin moisture, improving skin dryness and barrier damage caused by photoaging. Ectocin repairs "UV-induced cell damage," specifically by repairing fibroblast DNA and cell membrane damage caused by UV radiation, restoring the normal collagen synthesis function of fibroblasts; it also activates the skin cell's antioxidant system (such as the Nrf2 pathway), enhancing the skin's own resistance to UV radiation; in addition, it can downregulate ROS-mediated inflammatory factors such as IL-8, assisting gallic acid in reducing inflammatory responses and preventing the continued aggravation of collagen disorders.

[0163] A systematic study of photoaged skin revealed differences in the efficacy of different compositions and formulations in improving photoaged skin conditions. Based on comprehensive testing indicators, the efficacy ranking of the experimental groups was: Experimental Group 7 > Experimental Group 6 > Experimental Group 5 > Control Group > Experimental Group 4 > Experimental Group 3 > Experimental Group 1 > Experimental Group 2 > Model Group. Gallic acid or its derivatives, combined with silicone and ectoine, cover the entire chain of "UV protection → ROS scavenging → cell repair → collagen regulation." Through gallic acid's antioxidant and MMP-inhibiting effects, silicone's physical protection and collagen regulation, and ectoine's cell repair and enhanced resistance, the limitations of single-component formulations (such as vitamin E alone, which only has antioxidant properties but cannot regulate collagen arrangement) are overcome.

[0164] Experiments showed that experimental group 7 was superior to experimental groups 4 and 5 in improving skin fibrosis, inhibiting collagen deposition, and regulating fibroblast activation. This indicates that the combination of the three active substances produced a certain synergistic effect, which was better than the combination of any two substances alone. While gallic acid derivatives and silicones alone, as well as ectoine alone, have some effect on photoaging, their effects are far less than any combination of two substances, and far less than the effect of the combination of the three active substances.

[0165] The composition provided by this invention can significantly inhibit the process of collagen fibrosis through the synergistic effect of gallic acid derivatives, silicone, and ectoine. Experimental group 6 (a basic formula of gallic acid derivatives + polydimethylsiloxane + ectoine ointment) and experimental groups 7-3 (hydrocolloid formulations with the same composition) showed better effects, and the hydrocolloid formulation has certain advantages in photoaging treatment. This composition offers flexible dosage forms and high safety, providing a novel and efficient treatment option for collagen fibrosis.

[0166] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A composition capable of intervening in collagen fibrillation, characterized in that: This includes gallic acid or gallic acid derivatives, silicones, and ectoine.

2. The composition capable of intervening in collagen fibrosis according to claim 1, characterized in that: Calculated by mass fractions, it includes 0.01-10 parts gallic acid or gallic acid derivatives, 0.01-90 parts silicone, and 0.01-5 parts ectoine.

3. The composition capable of intervening in collagen fibrosis according to claim 1, characterized in that: Gallic acid derivatives are propyl gallate or bismuth subgallate.

4. The composition capable of intervening in collagen fibrosis according to claim 1, characterized in that: The silicone is polydimethylsiloxane, cyclopentadimethylsiloxane, or dimethyl silicone oil.

5. A formulation capable of intervening in collagen fibrosis, characterized in that: Includes the composition capable of intervening in collagen fibrillation as described in any one of claims 1-4.

6. The formulation capable of intervening in collagen fibrosis according to claim 5, characterized in that: The formulation is a cream or a hydrocolloid; the cream includes emulsions and ointments.

7. The formulation capable of intervening in collagen fibrosis according to claim 6, characterized in that: When the formulation is an emulsion, the composition comprises, by weight, 0.01-10 parts gallic acid or gallic acid derivatives and 20-70 parts silicone; or, the composition comprises 0.01-10 parts gallic acid or gallic acid derivatives, 20-70 parts silicone and 0.01-5 parts ectoine. When the formulation is a hydrocolloid, the composition comprises, by mass parts, 0.01-10 parts gallic acid or gallic acid derivatives and 0.01-10 parts silicone; or, the composition comprises 0.01-10 parts gallic acid or gallic acid derivatives, 0.01-10 parts silicone and 0.01-5 parts ectoine.

8. The formulation capable of intervening in collagen fibrosis according to claim 7, characterized in that: When the formulation is a cream, it also includes one or more of the following: excipients, emollients, thickeners, pH adjusters, preservatives, and water; when the formulation is an emulsion, it also includes an emulsifier; when the formulation is an ointment, it does not include an emulsifier. Alternatively, when the formulation is a hydrocolloid, it may also include one or more of the following: water-absorbing materials, adhesive substances, and plasticizers.

9. The use of the composition of claims 1-4 or the formulation of claims 5-8 in the preparation of products for treating collagen fibrosis diseases.

10. The application according to claim 9, characterized in that: Collagen fibrosis can manifest as scars, scleroderma, or photoaging of the skin.

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