A pharmaceutical preparation for repairing skin mucosa damage

Abstract

The present application relates to the technical field of skin mucosa repair, and particularly relates to a pharmaceutical preparation for repairing skin mucosa injury. The preparation takes kaempferol, quercetin, 3-p-coumaroyl-1, 4, 6'-triacetyl sucrose, 3-p-coumaroyl-2', 3', 6'-triacetyl sucrose, (+)-syringaresinol and vitamin B12 extracted from Prunus tomentosa as active ingredients, so as to solve the problems of poor curative effect, long repair period and easy scarring of the existing skin mucosa injury repair preparation, and realize efficient, safe and high-quality skin mucosa injury repair effect.

Description

Technical Field

[0001] This invention relates to the field of skin and mucous membrane repair technology, and in particular to a pharmaceutical preparation for repairing skin and mucous membrane damage. Background Technology

[0002] As the body's first line of defense against external aggressors, the integrity of the skin and mucous membranes is crucial for maintaining health. However, in daily life, the skin and mucous membranes are easily damaged by various factors such as burns, scalds, abrasions, surgical trauma, and diseases. Damaged skin and mucous membranes can not only cause pain and infection but also affect appearance and related physiological functions, and in severe cases, even threaten life and health.

[0003] Currently, there are numerous drug formulations on the market for repairing skin and mucous membrane injuries, but most suffer from problems such as poor efficacy, long repair cycles, and a tendency to leave scars. While some traditional drugs possess certain anti-inflammatory and anti-infective effects, their effectiveness is limited in key areas such as promoting cell proliferation, regulating collagen synthesis, and improving the structure and function of the skin and mucous membranes. For example, some common antibacterial ointments can only inhibit bacterial growth, with weak repair effects on damaged tissues; and while some moisturizing dressings can provide a certain level of hydration to the wound, they cannot fundamentally accelerate the regeneration and repair process of tissues. Therefore, developing a highly effective, safe drug formulation that can significantly improve the repair effect of skin and mucous membrane injuries is of significant clinical importance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a pharmaceutical formulation for repairing skin and mucous membrane damage. By innovatively utilizing a unique and rare combination of active ingredients from cherry blossoms, it solves the problems of poor efficacy, long repair cycles, and easy scarring found in existing skin and mucous membrane damage repair formulations. It demonstrates outstanding novelty and creativity in the field of skin and mucous membrane repair, achieving efficient, safe, and high-quality skin and mucous membrane damage repair effects.

[0005] Through in-depth research and screening using cutting-edge technologies, this invention precisely extracts active ingredients with superior skin and mucous membrane repair capabilities from wild cherry.

[0006] In a first aspect, the present invention provides a pharmaceutical preparation for repairing skin and mucous membrane damage, the preparation comprising a first *Prunus persica* extract, a second *Prunus persica* extract, a third *Prunus persica* extract, and vitamin B12. The first *Prunus persica* extract contains kaempferol and quercetin; the second *Prunus persica* extract contains 3-p-coumaryl-1,4,6'-triacetylsucrose and 3-p-coumaryl-2',3',6'-triacetylsucrose; and the third *Prunus persica* extract contains (+)-eugenol.

[0007] Among them, kaempferol (C 15 H10 O6) is a natural flavonol compound that exists in the form of free flavonols or glycosides in hairy cherries. Quercetin (C... 15 H 10 O7), also belonging to the flavonol class, possesses multiple phenolic hydroxyl structures. The chemical structure of 3-p-coumaryl-1,4,6'-triacetyl sucrose contains a p-coumaryl group linked to triacetyl sucrose, exhibiting unique physiological activities. 3-p-coumaryl-2',3',6'-triacetyl sucrose has a similar structure but differs in substituent positions. Previously, these extracts of *Prunus mume* were primarily reported for their antitumor activity; this invention reveals for the first time their unique efficacy in skin and mucous membrane repair.

[0008] (+)-Eucalyptus resinol, a lignan compound, exhibits significant advantages in promoting the proliferation of skin fibroblasts and collagen synthesis, accelerating the structural reconstruction and functional recovery of damaged skin and mucous membranes. Kaempferol effectively inhibits the release of key inflammatory factors such as IL-6 and TNF-α, reducing the damage of inflammation to skin and mucous membrane cells and creating an excellent repair microenvironment for damaged tissues. Quercetin possesses antioxidant activity, regulates cellular redox balance, efficiently scavenges excess intracellular free radicals, prevents oxidative damage to cell membranes, DNA, and proteins, and maintains normal cell structure and function. Extracts of *Prunus persica*, such as 3-p-coumaryl-1,4,6'-triacetyl sucrose and 3-p-coumaryl-2',3',6'-triacetyl sucrose, can regulate the expression of cell cycle-related proteins, such as upregulating the expression of cyclin D1, accelerating the proliferation and differentiation of skin fibroblasts, while simultaneously promoting the orderly synthesis and deposition of collagen and elastic fibers, enhancing the structural strength and elastic recovery capacity of skin and mucous membranes. (+)-Eucalyptus resin alcohol has significant advantages in promoting the proliferation of skin fibroblasts and collagen synthesis, and accelerates the structural reconstruction and functional recovery of damaged skin and mucous membranes.

[0009] This invention combines the above-mentioned active ingredients and adds appropriate pharmaceutical excipients to formulate ointments, gels, films, and other preparations. The specific types and proportions of excipients can be optimized and adjusted according to the type of preparation and actual needs.

[0010] In a second aspect, the present invention provides a gel for repairing skin and mucous membrane damage, comprising, by weight, 20‰-47‰ of first cherry extract, 3‰-5‰ of second cherry extract, 1‰-2‰ of third cherry extract, 0.1%-1% vitamin B12, 10‰-15‰ carbomer, 50‰-80‰ glycerin, 5‰-10‰ triethanolamine, and 1‰-2‰ methylparaben.

[0011] The preparation method of the gel includes: weighing carbomer, slowly sprinkling it into an appropriate amount of distilled water, stirring to allow it to swell fully, and obtaining a carbomer gel matrix. Dissolving the first, second, and third *Prunus persica* extracts separately in an appropriate amount of ethanol, then adding them to the carbomer gel matrix and stirring until homogeneous. Weighing glycerin, methylparaben, and vitamin B12, adding them to an appropriate amount of distilled water, heating and stirring to dissolve them, cooling, and then adding them to the above mixture. Slowly adding triethanolamine dropwise, adjusting the pH to 6.5-7.5, stirring continuously until a uniform and fine gel is formed.

[0012] In a third aspect, the present invention provides an ointment for repairing skin and mucous membrane damage, comprising, by weight, 30‰-50‰ of first cherry extract, 4‰-6‰ of second cherry extract, 1.5‰-2.5‰ of third cherry extract, 200‰-300‰ of petrolatum, 100‰-150‰ of lanolin, 50‰-100‰ of liquid paraffin, 0.1%-1% of vitamin B12, and 2‰-3‰ of ethylparaben.

[0013] The preparation method of the ointment includes: heating and melting petrolatum, lanolin, and liquid paraffin, stirring until homogeneous to obtain an oil phase. Dissolving first-harvest cherry extract, second-harvest cherry extract, third-harvest cherry extract, vitamin B12, and ethylparaben in an appropriate amount of ethanol, then adding the dissolved substances to the oil phase and stirring until homogeneous. Continuing to stir until cooled to room temperature, a homogeneous ointment is formed.

[0014] In some embodiments, the first *Prunus persica* extract contains an average percentage of 13.4% kaempferol and an average percentage of 18.6% quercetin by weight. The second *Prunus persica* extract contains an average percentage of 88.2% 3-p-coumaroyl-1,4,6'-triacetylsucrose and 3-p-coumaroyl-2',3',6'-triacetylsucrose by weight. The third *Prunus persica* extract contains 98.3% (+)-eugenol by weight.

[0015] In some embodiments, the preparation method of the first *Prunus persica* extract includes: taking dried *Prunus persica* fruit powder, adding 60% ethanol solution and refluxing for extraction. The extracts are combined and concentrated under reduced pressure until no alcohol odor remains, obtaining a concentrated solution. The concentrated solution is passed through a macroporous adsorption resin column and eluted sequentially with distilled water, 30% ethanol, and 70% ethanol. The 70% ethanol eluent is collected, concentrated under reduced pressure, and then freeze-dried to obtain the first *Prunus persica* extract rich in kaempferol and quercetin.

[0016] In some embodiments, the preparation method of the second *Prunus persica* extract includes: washing the residue after extracting the first *Prunus persica* extract with distilled water and drying it to constant weight; mixing the dried residue with distilled water at a solid-liquid ratio of 1:15, adjusting the pH, adding a compound enzyme, stirring evenly, and then transferring it to a constant temperature shaker for enzymatic hydrolysis, heating in an 80°C water bath for 15 minutes, cooling to room temperature, centrifuging at 4000 r / min for 10 minutes, and retaining the precipitate; wherein, the compound enzyme is a mixture of cellulase, pectinase, and xylanase; adding the precipitate after enzymatic hydrolysis to dilute sulfuric acid, heating in a water bath for 2 hours, adjusting the pH, centrifuging at 4000 r / min for 15 minutes, washing the precipitate with distilled water, and vacuum drying to obtain pretreated residue; adding the pretreated residue to an ethyl acetate-acetone mixed solvent and ultrasonically extracting it. The extracts were combined, concentrated under reduced pressure, and separated by silica gel column chromatography with petroleum ether-ethyl acetate gradient elution. The fractions were concentrated and dried to obtain the first powder. The first powder was dissolved in 50% methanol and loaded onto a reversed-phase C18 packed column. The column was eluted with a methanol-water gradient at a flow rate of 2 mL / min. The fractions with a purity >80% were collected, concentrated, and dried to obtain the second powder. The second powder was dissolved in an acetone-water mixture by heating, filtered while hot, and the filtrate was allowed to stand at 4°C for 24 hours to crystallize. The crystals were filtered to obtain white needle-like crystals, which were then dried under vacuum to obtain the second extract of Prunus persica.

[0017] In some embodiments, the preparation method of the third *Prunus persica* extract includes: washing the remaining residue after extracting the second *Prunus persica* extract with n-hexane and drying it under vacuum; extracting the remaining residue with methanol-water under reflux. Combining the extracts, concentrating under reduced pressure, and extracting three times with an equal volume of ethyl acetate, combining the ethyl acetate phases, concentrating under reduced pressure to dryness to obtain the third powder; dissolving the third powder in methanol and separating it on an ODS column using methanol-water gradient elution, detecting it by HPLC, combining the fractions containing (+)-eugenol, concentrating and drying to obtain the fourth powder; dissolving the fourth powder in a mobile phase of methanol-0.1% formic acid-water and loading it onto HPLC, collecting the (+)-eugenol main peak, combining the collected liquids, concentrating under reduced pressure to dryness to obtain the fifth powder; dissolving the fifth powder in a methanol-petroleum ether mixed solvent by heating, allowing it to stand at room temperature for 12 hours to precipitate white needle-like crystals, filtering and drying under vacuum at 40°C to obtain the third *Prunus persica* extract.

[0018] This invention also aims to provide a membrane preparation for skin and mucous membrane repair, which utilizes the unique combination of active ingredients in cherry blossoms to solve the problems of low repair efficiency and poor biocompatibility of existing skin and mucous membrane repair materials, thereby achieving efficient repair of skin and mucous membrane damage.

[0019] In a fourth aspect, the present invention provides a membrane preparation for repairing skin and mucous membrane damage, comprising, by weight, 10% to 25% of first-fleshed cherry extract, 1% to 3% of second-fleshed cherry extract, 0.5% to 1.5% of third-fleshed cherry extract, 0.1% to 1% vitamin B12, 20% to 30% chitosan, 10% to 20% gelatin, 0.1% to 2% tannic acid, and the balance being water.

[0020] The preparation method of the membrane formulation includes: dissolving vitamin B12, chitosan, and gelatin in a 1% dilute acetic acid solution (solid-liquid ratio 1:10) at the above ratio, stirring at 37°C for 1 hour until completely dissolved to obtain a substrate solution. Adding first, second, and third *Prunus persica* extracts sequentially to the substrate solution, stirring at 30°C for 30 minutes until homogeneous. Then adding tannic acid and continuing stirring for 40 minutes, pouring the mixture into a polytetrafluoroethylene mold (thickness controlled at 0.2mm-0.3mm), drying in a 37°C oven for 24 hours to form the membrane, and demolding to obtain a membrane formulation for skin and mucous membrane repair.

[0021] In summary, the beneficial effects of this invention are as follows:

[0022] The pharmaceutical formulation of this invention exhibits significant advantages in reducing the incidence of scar hyperplasia, substantially shortening the wound healing cycle, and improving the quality of skin and mucous membrane repair through the synergistic effect of multiple active ingredients. Experimental verification shows that, compared with existing technologies, the wound healing time using the formulation of this invention can be shortened by 35%, and the incidence of scar hyperplasia can be reduced by 40%.

[0023] This invention reveals for the first time the unique mechanism of action of various active ingredients in Prunus persica in skin and mucous membrane repair, especially the new application of Prunus persica extract in the field of skin and mucous membrane repair, providing a brand-new idea and direction for the development of drugs for skin and mucous membrane damage repair.

[0024] As a natural plant, the active ingredients of hairy cherry are safe and reliable. Furthermore, the selection and use of excipients are strictly controlled during the formulation process to avoid the use of substances harmful to the human body, thus ensuring the safety of the formulation and reducing the risk of adverse reactions.

[0025] The membrane formulation provided by this invention contains multiple active ingredients that work synergistically to effectively promote the proliferation and differentiation of skin and mucous membrane cells and accelerate the repair process of damaged mucous membranes.

[0026] This invention uses natural biodegradable polymer materials as the base material, which has good biocompatibility, will not cause adverse gastrointestinal reactions, and can gradually degrade during the repair process without the need for secondary removal.

[0027] The membrane formulation provided by this invention has certain mechanical strength and barrier function, which can protect damaged skin and mucous membranes from further irritation and damage, and provide a good microenvironment for mucous membrane repair. Attached Figure Description

[0028] Figure 1 The cell proliferation rates of Example 2, Comparative Example 1, Comparative Example 4, Comparative Example 7 and the blank group in the gel cell experiment are shown. "*" indicates p<0.05 compared with Example 2, "**" indicates p<0.01 compared with Example 2, and "***" indicates p<0.005 compared with Example 2.

[0029] Figure 2 The values ​​of IL-6 and TNF-α in Example 2, Comparative Example 1, Comparative Example 4, Comparative Example 7 and the blank group in the gel cell experiment are shown. "*" indicates p < 0.05 compared with Example 2, "**" indicates p < 0.01 compared with Example 2, and "***" indicates p < 0.005 compared with Example 2.

[0030] Figure 3 The wound healing time of Examples 2, 3, Comparative Examples 1, 2, 4, 5, 7, 8 and the blank group in animal experiments on skin and mucous membrane injury repair is given. "*" indicates p<0.05 compared with Example 2 or Example 3, and "**" indicates p<0.01 compared with Example 2 or Example 3.

[0031] Figure 4 The collagen content in the wound tissues of Examples 2, 3, Comparative Examples 1, 2, 4, 5, 7, 8 and the blank group in animal experiments on skin and mucous membrane injury repair is shown. "*" indicates p<0.05 compared with Example 2 or Example 3, and "**" indicates p<0.01 compared with Example 2 or Example 3.

[0032] Figure 5 The relative cell proliferation rates of Example 4, Comparative Example 3, Comparative Example 6, Comparative Example 9 and the blank group in the in vitro experiments of the membrane formulation are given. "ns" indicates no significant difference.

[0033] Figure 6 The cell proliferation rates at 24h, 48h, and 72h for Example 4, Comparative Example 3, Comparative Example 6, Comparative Example 9, and the blank group in the in vitro experiments of the membrane formulation are shown. "*" indicates p<0.05 compared with Example 4, "**" indicates p<0.01 compared with Example 4, and "***" indicates p<0.005 compared with Example 4.

[0034] Figure 7The values ​​for IL-6, TNF-α, and collagen content in Example 4, Comparative Example 3, Comparative Example 6, and Comparative Example 9 in in vitro experiments of the membrane formulation are shown. "*" indicates p < 0.05 compared to Example 4, "**" indicates p < 0.01 compared to Example 4, and "***" indicates p < 0.005 compared to Example 4.

[0035] Figure 8 The wound healing rates at 24 h and 48 h for Examples 4, 3, 6 and 9 in the in vitro experiments of the membrane formulation are shown. "*" indicates p < 0.05 compared with Example 4, "**" indicates p < 0.01 compared with Example 4, and "***" indicates p < 0.005 compared with Example 4. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents not specifically described in detail in this invention are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0037] Example 1: Preparation of active ingredient extract from Prunus persica:

[0038] 1. First cherry extract:

[0039] 1000g of dried Prunus persica fruit powder was added to 8 times its volume of 60% ethanol solution and extracted three times by reflux at 60℃, 1.5 hours each time. The extracts were combined and concentrated under reduced pressure until no alcohol odor remained, yielding a concentrated solution. The concentrated solution was passed through a macroporous adsorption resin column and eluted sequentially with distilled water, 30% ethanol, and 70% ethanol. The 70% ethanol eluent was collected, concentrated under reduced pressure, and then freeze-dried to obtain a Prunus persica extract rich in kaempferol and quercetin. Analysis showed that the Prunus persica extract contained 13.4% kaempferol and 18.6% quercetin.

[0040] 2. Second cherry extract:

[0041] The residue after extracting the first cherry extract was washed with distilled water and dried to constant weight.

[0042] The dried medicinal residue was mixed with distilled water at a solid-liquid ratio of 1:15. The pH was adjusted to 4.5-5.0 with 0.1 mol / L citrate-sodium citrate buffer. The compound enzyme was added, and the mixture was stirred evenly. The mixture was then transferred to a 50°C constant temperature shaker and enzymatically hydrolyzed at 150 rpm for 3 hours. After hydrolysis, the mixture was heated in an 80°C water bath for 15 minutes, cooled to room temperature, and centrifuged at 4000 rpm for 10 minutes. The precipitate was retained. The compound enzyme was a mixture of cellulase with an activity ≥5000 U / g, pectinase with an activity ≥3000 U / g, and xylanase with an activity ≥2000 U / g, in a mass ratio of 3:2:1.

[0043] The enzymatically hydrolyzed precipitate was added to 0.3% (v / v) dilute sulfuric acid at a solid-liquid ratio of 1:15. The mixture was heated in a water bath at 50°C for 2 hours, with stirring every 30 minutes. The pH was adjusted to 6.5-7.0 with 10% barium hydroxide solution. The mixture was centrifuged at 4000 r / min for 15 minutes. The precipitate was washed with distilled water and dried under vacuum at 60°C to obtain the pretreated residue.

[0044] The pretreated residue was added to 8 times its volume of ethyl acetate-acetone mixed solvent (9:1) and extracted four times by ultrasonication (300W) at 50℃ for 40 minutes each time. The extracts were combined, concentrated under reduced pressure, and then separated by silica gel column chromatography (200-300 mesh silica gel, column volume (Φ5cm×50cm)) using a petroleum ether-ethyl acetate gradient elution (5:1→3:1→1:1). One fraction was collected every 500mL and detected by TLC (developing solvent: chloroform-methanol=8:2, UV 254nm). The fractions containing the target component (mainly the 3:1 elution fraction) were combined, concentrated, and dried to obtain the first powder.

[0045] The first powder was dissolved in 50% methanol and loaded onto a reversed-phase C18 packed column (5 μm, column volume (Φ3cm×30cm)). Methanol-water gradient elution (30%→50%→70%) was performed at a flow rate of 2 mL / min. HPLC detection was performed (C18 column, mobile phase methanol-0.1% phosphoric acid water = 45:55, detection wavelength 320 nm). The fraction with a purity >80% was collected, concentrated, and dried to obtain the second powder.

[0046] The obtained second powder was dissolved in an acetone-water (7:3) mixture at 60°C upon heating, filtered while hot, and the filtrate was allowed to stand at 4°C for 24 hours to crystallize. The resulting white needle-like crystals were filtered and dried under vacuum at 60°C to obtain the second hairy cherry extract. Extraction and HPLC analysis showed that the total content of 3-p-coumaryl-1,4,6'-triacetylsucrose and 3-p-coumaryl-2',3',6'-triacetylsucrose in the second hairy cherry extract was 88.2%.

[0047] 3. Third hairy cherry extract:

[0048] The remaining residue after extracting the second cherry extract was washed with hexane and then vacuum dried at 60°C.

[0049] The remaining residue after pretreatment was extracted three times by reflux with 10 times the volume of methanol-water (8:2) at 65°C for 2 hours each time. The extracts were combined, concentrated under reduced pressure, and then extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated to dryness under reduced pressure at 50°C to obtain the third powder.

[0050] The third powder was dissolved in 70% methanol and loaded onto an ODS column (10 μm, column volume (Φ3cm×30cm)) for chromatographic separation. The column was eluted with a methanol-water gradient (60%→80%→100%) at a flow rate of 2 mL / min. The sample was detected by HPLC (C18 column, mobile phase methanol-water = 70:30, detection wavelength 280 nm). The fractions containing (+)-eugenol (80% methanol elution fraction) were combined, concentrated, and dried to obtain the fourth powder (approximately 1-1.5 g).

[0051] The fourth powder was dissolved in a mobile phase of methanol-0.1% formic acid water = 65:35 and then loaded onto a preparative HPLC system (C18 preparative column, 250 mm × 20 mm, 5 μm) at a flow rate of 10 mL / min and a detection wavelength of 280 nm. The (+)-eugenol main peak was collected based on the peak area (retention time approximately 12.5 min). The collected solutions were combined and concentrated to dryness under reduced pressure at 50 °C to obtain the fifth powder.

[0052] The fifth powder was dissolved in a methanol-petroleum ether (1:3) mixed solvent and heated to 60°C. After standing at room temperature for 12 hours, white needle-like crystals precipitated. After filtration, the crystals were dried under vacuum at 40°C to obtain the third hairy cherry extract.

[0053] Extraction and liquid chromatography analysis (area normalization method) revealed that the content of (+)-eugenol in the extract of Cherry ternata was 98.3%.

[0054] Example 2: Preparation of a gel for repairing skin and mucous membrane damage:

[0055] 1. Gel formulation:

[0056] The gel formula (1000g) for repairing skin and mucous membrane damage provided in Example 2 is as follows: 35g of first cherry extract, 4g of second cherry extract, 1.5g of third cherry extract, 5g of vitamin B12, 12g of carbomer, 60g of glycerin, 8g of triethanolamine, 1.5g of methylparaben, and the remainder of distilled water.

[0057] 2. Preparation method of gelling agent:

[0058] The preparation method of the gel for repairing skin and mucous membrane damage provided in Example 2 is as follows:

[0059] Weigh out carbomer, slowly sprinkle it into an appropriate amount of distilled water, and stir to allow it to swell fully, thus obtaining a carbomer gel matrix.

[0060] The first, second, and third cherry extracts were dissolved in an appropriate amount of ethanol and then added to the carbomer gel matrix and stirred until homogeneous.

[0061] Weigh out glycerin, methylparaben, and vitamin B12, add them to an appropriate amount of distilled water, heat and stir to dissolve them, and then add them to the above mixture after cooling.

[0062] Slowly add triethanolamine dropwise, adjusting the pH to 6.5-7.5 while stirring, until a uniform and fine gel is formed.

[0063] Example 3: Preparation of an ointment for repairing skin and mucous membrane damage:

[0064] 1. Ointment formulation:

[0065] The ointment (1000g) for repairing skin and mucous membrane damage provided in Example 3 is formulated as follows: 48g of first cherry extract, 5g of second cherry extract, 2g of third cherry extract, 5g of vitamin B12, 250g of petrolatum, 120g of lanolin, 80g of liquid paraffin, 2.5g of ethylparaben, and the balance of ethanol.

[0066] 2. Preparation method of ointment:

[0067] The preparation method of the ointment for repairing skin and mucous membrane damage provided in Example 3 is as follows:

[0068] Petrolatum, lanolin, and liquid paraffin are heated and melted, and stirred evenly to obtain the oil phase.

[0069] The extracts of first-grade cherry, second-grade cherry, third-grade cherry, vitamin B12, and ethylparaben were dissolved in an appropriate amount of ethanol and then added to the oil phase and stirred until homogeneous.

[0070] Continue stirring until cooled to room temperature to form a uniform paste.

[0071] Example 4: Preparation of a membrane formulation for repairing skin and mucous membrane damage:

[0072] 1. Composition of the membrane formulation:

[0073] Example 4 describes a membrane formulation for repairing skin and mucous membrane damage. It uses biodegradable natural polymers (such as chitosan and gelatin) as a base material and loads active ingredients from *Prunus persica*, including first *Prunus persica* extract, second *Prunus persica* extract, third *Prunus persica* extract, and vitamin B12. Specifically, each 100g membrane formulation contains 17g of first *Prunus persica* extract, 2.0g of second *Prunus persica* extract, 0.8g of third *Prunus persica* extract, 0.5g of vitamin B12, 25g of chitosan, 16.5g of gelatin, and 0.6g of tannic acid, with the remainder being deionized water.

[0074] 2. Preparation method of membrane formulation:

[0075] Vitamin B12, chitosan, and gelatin were dissolved in a 1% dilute acetic acid solution according to the film formulation formula. The solution was stirred at 37°C for 1 hour until completely dissolved to obtain a substrate solution. The active ingredient of *Prunus persica* was added sequentially to the substrate solution, and the mixture was stirred at 30°C for 30 minutes until homogeneous. Tannic acid was then added, and stirring continued for 40 minutes. The mixture was poured into a polytetrafluoroethylene mold (thickness controlled at 0.2 mm - 0.3 mm), dried in a 37°C oven for 24 hours to form the film. After demolding, a film formulation for skin and mucous membrane repair was obtained.

[0076] Comparative Example 1: Preparation of a gel for repairing skin and mucous membrane damage:

[0077] The gel formulation (1000g) for repairing skin and mucous membrane damage provided in Comparative Example 1 is as follows: 40.5g of first cherry extract, 5g of vitamin B12, 12g of carbomer, 60g of glycerin, 8g of triethanolamine, 1.5g of methylparaben, and the remainder being distilled water. Its preparation method is the same as in Example 2.

[0078] Comparative Example 2: Preparation of an ointment for repairing skin and mucous membrane damage:

[0079] The formulation for the ointment (1000g) used to repair skin and mucous membrane damage provided in Comparative Example 2 is as follows: 55g of first cherry extract, 5g of vitamin B12, 250g of petrolatum, 120g of lanolin, 80g of liquid paraffin, 2.5g of ethylparaben, and the balance being ethanol. The preparation method is the same as in Example 3.

[0080] Comparative Example 3: Preparation of a membrane formulation for repairing skin and mucous membrane damage:

[0081] The formulation (100g) of the membrane preparation for repairing skin and mucous membrane damage provided in Comparative Example 3 is as follows: 19.8g of first cherry extract, 0.5g of vitamin B12, 25g of chitosan, 16.5g of gelatin, and 0.6g of tannic acid, with the remainder being deionized water. Its preparation method is the same as in Example 4.

[0082] Comparative Example 4: Preparation of a gel for repairing skin and mucous membrane damage:

[0083] The formula for the gel (1000g) used to repair skin and mucous membrane damage provided in Comparative Example 4 is as follows: 40.5g of *Prunus persica* extract, 5g of vitamin B12, 12g of carbomer, 60g of glycerin, 8g of triethanolamine, 1.5g of methylparaben, and the remainder being distilled water. Its preparation method is the same as in Example 2.

[0084] Comparative Example 5: Preparation of an ointment for repairing skin and mucous membrane damage:

[0085] The formulation for the ointment (1000g) used to repair skin and mucous membrane damage provided in Comparative Example 5 is as follows: 55g of second cherry extract, 5g of vitamin B12, 250g of petrolatum, 120g of lanolin, 80g of liquid paraffin, 2.5g of ethylparaben, and the balance being ethanol. The preparation method is the same as in Example 3.

[0086] Comparative Example 6: Preparation of a membrane formulation for repairing skin and mucous membrane damage:

[0087] The formulation (100g) of the membrane preparation for repairing skin and mucous membrane damage provided in Comparative Example 6 is as follows: 19.8g of *Prunus persica* extract, 0.5g of vitamin B12, 25g of chitosan, 16.5g of gelatin, and 0.6g of tannic acid, with the remainder being deionized water. Its preparation method is the same as in Example 4.

[0088] Comparative Example 7: Preparation of a gel for repairing skin and mucous membrane damage:

[0089] The formula for the gel (1000g) used to repair skin and mucous membrane damage provided in Comparative Example 7 is as follows: 40.5g of cherry amaranth extract, 5g of vitamin B12, 12g of carbomer, 60g of glycerin, 8g of triethanolamine, 1.5g of methylparaben, and the remainder being distilled water. Its preparation method is the same as in Example 2.

[0090] Comparative Example 8: Preparation of an ointment for repairing skin and mucous membrane damage:

[0091] The formulation for the ointment (1000g) used to repair skin and mucous membrane damage provided in Comparative Example 8 is as follows: 55g of cherry amaranth extract, 5g of vitamin B12, 250g of petrolatum, 120g of lanolin, 80g of liquid paraffin, 2.5g of ethylparaben, and the balance being ethanol. The preparation method is the same as in Example 3.

[0092] Comparative Example 9: Preparation of a membrane formulation for repairing skin and mucous membrane damage:

[0093] The formulation (100g) of the membrane preparation for repairing skin and mucous membrane damage provided in Comparative Example 9 is as follows: 19.8g of Cherry amaranth extract, 0.5g of vitamin B12, 25g of chitosan, 16.5g of gelatin, and 0.6g of tannic acid, with the remainder being deionized water. Its preparation method is the same as in Example 4.

[0094] I. Gel-based cell experiments:

[0095] 1. Experimental materials:

[0096] Cell experiments were conducted using the gels provided in Example 2, Comparative Example 1, Comparative Example 4, and Comparative Example 7, respectively.

[0097] Cell culture: Human skin fibroblasts were taken and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were used for experiments when they grew to the logarithmic growth phase.

[0098] 2. Group processing:

[0099] Cells were divided into experimental and control groups. The experimental groups were given culture media containing gels provided in Example 2 at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL; gels provided in Comparative Example 1 at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL; gels provided in Comparative Example 4 at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL; and gels provided in Comparative Example 7 at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL. The control group was given culture media without drugs.

[0100] 3. Cell proliferation detection (CCK-8 assay):

[0101] After culturing the treated cells for 72 hours, 10 μL of LCK-8 solution was added to each well, and the cells were cultured for another 2 hours. The absorbance was then measured at 450 nm using a microplate reader.

[0102] like Figure 1 As shown, at all concentrations, the cell proliferation rate of Example 2 was significantly higher than that of all comparative examples (P<0.05). Comparative Example 7 (containing only the third cherry extract) was superior to Comparative Example 4 (containing only the second cherry extract) and Comparative Example 1 (containing only the first cherry extract), indicating that (+)-eugenol has a stronger effect on promoting fibroblast proliferation; while Example 2 showed the best proliferation effect due to the synergistic effect of the three extracts and vitamin B12 (vitamin B12 promotes cell metabolism and enhances fibroblast activity).

[0103] 4. Inflammatory factor detection (ELISA method):

[0104] like Figure 2 As shown, the IL-6 and TNF-α levels in Example 2 were significantly lower than those in all comparative examples (P<0.05). Comparative Example 1 (only the extract of *Prunus persica*) showed better anti-inflammatory effects than Comparative Examples 4 and 7 because kaempferol and quercetin are the main anti-inflammatory components. In Example 2, vitamin B12 downregulated inflammatory signaling pathways, synergistically reducing the levels of inflammatory factors and providing a better microenvironment for wound repair, in conjunction with the anti-inflammatory effect of *Prunus persica* extract.

[0105] II. Animal experiments verifying the efficacy of the formulation in repairing skin and mucous membrane injuries:

[0106] 1. Laboratory animals and grouping:

[0107] Sixty healthy adult SD rats were randomly divided into four groups: Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 4, Comparative Example 5, Comparative Example 7, Comparative Example 8, and a blank group (using blank matrix), with 20 rats in each group.

[0108] 2. Modeling method:

[0109] A circular skin lesion with a diameter of 2 cm was created on the back of a rat.

[0110] 3. Administration method:

[0111] Each day, the corresponding drug was applied to the skin lesions of rats in each group. For example, the gel was applied to the rats in Example 2, and the ointment was applied to the rats in Example 3. The gel was 0.5g and the ointment was 0.2g. The administration was carried out continuously for 14 days.

[0112] 4. Wound healing time:

[0113] like Figure 3 As shown, the healing time of Examples 2 and 3 was significantly shorter than that of all comparative examples (P<0.05). Comparative examples 7-8 (third cherry extract only) healed faster than comparative examples 4-5 (second cherry extract only) and 1-2 (first cherry extract only); while in Examples 2-3, the three extracts worked synergistically with vitamin B12, increasing the healing efficiency by 30%-40%.

[0114] 5. Collagen synthesis detection:

[0115] Wound tissues from Example 2 group on day 8, Example 3 group on day 8, Comparative Example 1 group on day 12, and Comparative Example 2 group on day 12 were collected respectively, and the collagen content was detected.

[0116] like Figure 4As shown, the collagen content of Examples 2-3 was significantly higher than that of all comparative examples (P<0.05). Comparative examples 7-8 (only the extract of Cherry ternata) had higher collagen content than other comparative examples because (+)-eugenol directly promoted collagen synthesis; while in Examples 2-3, the sucrose derivative of the Cherry ternata extract regulated the orderly deposition of collagen, and vitamin B12 enhanced fibroblast activity, which synergistically improved the total amount and quality of collagen with the Cherry ternata extract, thus accelerating the reconstruction of wound structure.

[0117] III. In vitro experiments using membrane formulations:

[0118] 1. Experimental materials:

[0119] Membrane preparations: The membrane preparations for skin and mucous membrane repair prepared in Example 4, Comparative Example 3, Comparative Example 6 and Comparative Example 9 were cut into circular samples with a diameter of 1 cm.

[0120] Cells: a single layer of columnar epithelial cells.

[0121] Culture medium: DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0122] Main reagents: CCK-8 kit, IL-6 and TNF-α ELISA kit, collagen detection kit, etc.

[0123] 2. Experimental methods:

[0124] Preparation of membrane formulation extracts: In accordance with ISO10993-12 standard, the membrane formulations of Example 4 and Comparative Example 3 were placed in centrifuge tubes, and DMEM / F12 medium containing 10% fetal bovine serum (material to medium volume ratio of 1:10) was added. The extracts were then extracted in a 37°C, 5% CO2 incubator for 24 h. Subsequently, the extracts were filtered through a 0.22 μm filter membrane for sterilization to obtain extracts of the two membrane formulations for later use.

[0125] Cytotoxicity assay (CCK-8 assay): Logarithmic growth phase monolayer columnar epithelial cells were digested with trypsin-EDTA digestion solution, and then 5 × 10⁶ cells were injected into each well. 3Cells were seeded at a density of 100 μL of culture medium per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. The old culture medium was discarded, and extracts of the membrane formulation from Example 4 (Example 4 group) and Comparative Example 3 (Comparative Example 3 group) were added, along with a control group (containing an equal volume of fresh culture medium). Each group had 6 replicates, with 100 μL per well. After 24 h, 48 h, and 72 h of incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2 h. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader, and the relative cell proliferation rate was calculated as follows: Relative cell proliferation rate (%) = (OD value of experimental group / OD value of control group) × 100%.

[0126] Cell proliferation assay: Monolayer columnar epithelial cells were seeded into 96-well plates. After cell adhesion, the membrane preparation extract of Example 4 (Example 4 group), the membrane preparation extract of Comparative Example 3 (Comparative Example 3 group), and drug-free culture medium (blank group) were added respectively. After culturing for 24h, 48h, and 72h, cell proliferation was detected by CCK-8 assay, and the cell proliferation rate was calculated as follows: Cell proliferation rate (%) = (OD value of experimental group / OD value of blank group) × 100%.

[0127] Inflammatory factor detection (ELISA method): monolayer columnar epithelial cells were sampled at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in a 24-well plate, with 500 μL of culture medium added to each well. After 24 h of culture, the old culture medium was discarded, and the membrane formulation extracts from Example 4, Comparative Example 3, and fresh culture medium (blank control) were added to each well, with three replicates per group. After 48 h of culture, the cell supernatant from each well was collected, centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatant was collected. The levels of IL-6 and TNF-α in the supernatant were detected by ELISA.

[0128] Collagen synthesis detection: Cell seeding and grouping were the same as in the inflammatory factor detection experiment. After culturing for 72 h, the culture medium was discarded, and the cells were washed twice with PBS. 200 μL of trypsin-EDTA digestion solution was added to each well, and the cells were incubated at 37°C for 5 min. Cells were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and 100 μL of cell lysis buffer was added. The cells were incubated on ice for 30 min, vortexing three times for 10 s each time. After centrifugation at 4°C and 12000 rpm for 15 min, the supernatant was collected, and the collagen content was detected according to the collagen detection kit instructions. Simultaneously, the protein concentration was determined using the BCA method, and the collagen content was standardized to the amount of collagen per mg of total protein.

[0129] In vitro wound healing experiment: monolayer columnar epithelial cells were cultured at 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 μL per well in 6-well plates, with 2 mL of culture medium added to each well. Cells were cultured until confluence reached 90% or higher. Using a sterile 10 μL pipette tip, a straight line was drawn perpendicularly to the surface of each well to simulate a skin or mucous membrane injury. The initial width of the scratch was measured beforehand using Image-ProPlus, and wells with a difference of <10% were selected for the experiment. The plates were gently rinsed three times with PBS buffer to remove the drawn cells. Then, the membrane preparation extracts from Example 4 (Example 4 group), Comparative Example 3 (Comparative Example 3 group), and a blank group (with an equal volume of fresh culture medium) were added, with three replicates per group. Immediately, observation points were marked and fixed on both sides of the scratch under an inverted microscope (100×magnification), and images of the wound were taken at 0 h. The 6-well plates were then placed in a 37°C, 5% CO2 incubator for further culture, and images of the wound were taken at the same observation points at 24 h and 48 h. Image-ProPlus 6.0 image analysis software was used to measure the width of the wound at each time point, and the wound healing rate was calculated: Wound healing rate (%) = (0h wound width - wound width at each time point) / 0h wound width × 100%.

[0130] 3. Experimental Results:

[0131] like Figure 5 As shown, the relative cell proliferation rate of all membrane preparations was >90% (ISO10993-5 standard), and there was no cytotoxicity, indicating that the combination of vitamin B12 with the three extracts has good biocompatibility.

[0132] like Figure 6 As shown, the proliferation rate of monolayer columnar epithelial cells in Example 4 was significantly higher than that in all comparative examples (P<0.05). Vitamin B12 in Example 4 promotes DNA synthesis in mucosal cells and, synergistically with the extracts of second and third *Prunus persica* (which regulate the cell cycle) and promote proliferation, accelerates mucosal cell regeneration.

[0133] like Figure 7 As shown, Example 4 exhibited the lowest inflammatory factor content and the highest collagen content (P<0.05). This indicates that the membrane preparation provided by the present invention, through the synergistic effect of the first, second, and third *Prunus persica* extracts, not only has anti-inflammatory properties but also promotes collagen synthesis in skin and mucous membrane cells, which is beneficial for mucosal repair and regeneration.

[0134] like Figure 8 As shown, the wound healing rate of Example 4 was significantly higher than that of all comparative examples (P<0.05), with a healing rate of 85.3% after 48 hours. This indicates that the three extracts and vitamin B12 synergistically promote mucosal cell migration and wound closure. In particular, vitamin B12 can enhance cell vitality and accelerate the coverage of the damaged area.

[0135] In summary, this invention, through the synergistic effect of the first, second, and third *Prunus persica* extracts, demonstrates significantly superior performance compared to single-extract groups in terms of cell proliferation, anti-inflammation, wound healing, and collagen synthesis. Furthermore, this invention, through the synergistic effect of the first, second, and third *Prunus persica* extracts and vitamin B12, participates in cellular DNA synthesis and energy metabolism, enhancing the activity of the three extracts and further shortening the repair cycle and improving repair quality.

[0136] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A gel for repairing skin and mucous membrane damage, characterized in that, The gel comprises, by weight, 20‰-47‰ of first hairy cherry extract, 3‰-5‰ of second hairy cherry extract, 1‰-2‰ of third hairy cherry extract, 0.1%-1% vitamin B12, 10‰-15‰ carbomer, 50‰-80‰ glycerin, 5‰-10‰ triethanolamine, and 1‰-2‰ methylparaben. The first hairy cherry extract contains kaempferol and quercetin; the second hairy cherry extract contains 3-p-coumaryl-1,4,6'-triacetylsucrose and 3-p-coumaryl-2',3',6'-triacetylsucrose; and the third hairy cherry extract contains (+)-eugenol. The preparation method of the first hairy cherry extract includes: Take dried Prunus pubescens fruit powder, add 60% ethanol solution and reflux to extract; combine the extracts and concentrate under reduced pressure until there is no alcohol odor to obtain a concentrated solution; pass the concentrated solution through a macroporous adsorption resin column and elute with distilled water, 30% ethanol and 70% ethanol in sequence, collect the 70% ethanol eluent, concentrate under reduced pressure and freeze dry to obtain the first Prunus pubescens extract containing kaempferol and quercetin. The preparation method of the second hairy cherry extract includes: The residue after extracting the first cherry extract was washed with distilled water and dried to constant weight. The dried residue was mixed with distilled water at a solid-liquid ratio of 1:

15. After adjusting the pH, a compound enzyme was added, stirred evenly, and then transferred to a constant temperature shaker for enzymatic hydrolysis. The mixture was heated in an 80°C water bath for 15 minutes, cooled to room temperature, and centrifuged at 4000 r / min for 10 minutes. The precipitate was retained. The compound enzyme was a mixture of cellulase, pectinase, and xylanase. The precipitate after enzymatic hydrolysis was added to dilute sulfuric acid, heated in a water bath for 2 hours, the pH was adjusted, centrifuged at 4000 r / min for 15 minutes, the precipitate was washed with distilled water and vacuum dried to obtain pretreated drug residue. The pretreated residue was added to an ethyl acetate-acetone mixed solvent and extracted by ultrasonication; the extracts were combined, concentrated under reduced pressure, and then separated by silica gel column chromatography with petroleum ether-ethyl acetate gradient elution, concentrated and dried to obtain the first powder. The first powder was dissolved in 50% methanol and loaded onto a reversed-phase C18 packed column. The column was eluted with a methanol-water gradient at a flow rate of 2 mL / min. The fraction with a purity >80% was collected, concentrated, and dried to obtain the second powder. The obtained second powder was dissolved in acetone-water mixed solvent by heating, filtered while hot, and the filtrate was allowed to stand at 4°C for 24 hours to crystallize. After filtration, white needle-like crystals were obtained and dried under vacuum to obtain the second hairy cherry extract. The preparation method of the third hairy cherry extract includes: The remaining residue after extracting the second cherry extract was washed with hexane and then vacuum dried. The remaining residue was extracted by reflux with methanol-water, the extracts were combined, concentrated under reduced pressure, and then extracted multiple times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to dryness to obtain the third powder. The third powder was dissolved in methanol and separated by ODS column chromatography with methanol-water gradient elution and HPLC detection. The fractions containing (+)-eugenol were combined, concentrated and dried to obtain the fourth powder. The fourth powder was dissolved in the mobile phase of methanol-0.1% formic acid water and then loaded onto HPLC. The main peak of (+)-eugenol was collected. The collected liquids were combined and concentrated to dryness under reduced pressure to obtain the fifth powder. The fifth powder was dissolved in a methanol-petroleum ether mixture by heating, and allowed to stand at room temperature for 12 hours to precipitate white needle-like crystals. After filtration, the crystals were dried under vacuum at 40°C to obtain the third hairy cherry extract.

2. A cream for repairing skin and mucous membrane damage, characterized in that, The ointment comprises, by weight, 30‰-50‰ of first-hair cherry extract, 4‰-6‰ of second-hair cherry extract, 1.5‰-2.5‰ of third-hair cherry extract, 200‰-300‰ of petrolatum, 100‰-150‰ of lanolin, 50‰-100‰ of liquid paraffin, 0.1%-1% of vitamin B12, and 2‰-3‰ of ethylparaben; The first hairy cherry extract contains kaempferol and quercetin; the second hairy cherry extract contains 3-p-coumaryl-1,4,6'-triacetylsucrose and 3-p-coumaryl-2',3',6'-triacetylsucrose; and the third hairy cherry extract contains (+)-eugenol. The preparation method of the first hairy cherry extract includes: Take dried Prunus pubescens fruit powder, add 60% ethanol solution and reflux to extract; combine the extracts and concentrate under reduced pressure until there is no alcohol odor to obtain a concentrated solution; pass the concentrated solution through a macroporous adsorption resin column and elute with distilled water, 30% ethanol and 70% ethanol in sequence, collect the 70% ethanol eluent, concentrate under reduced pressure and freeze dry to obtain the first Prunus pubescens extract containing kaempferol and quercetin. The preparation method of the second hairy cherry extract includes: The residue after extracting the first cherry extract was washed with distilled water and dried to constant weight. The dried residue was mixed with distilled water at a solid-liquid ratio of 1:

15. After adjusting the pH, a compound enzyme was added, stirred evenly, and then transferred to a constant temperature shaker for enzymatic hydrolysis. The mixture was heated in an 80°C water bath for 15 minutes, cooled to room temperature, and centrifuged at 4000 r / min for 10 minutes. The precipitate was retained. The compound enzyme was a mixture of cellulase, pectinase, and xylanase. The precipitate after enzymatic hydrolysis was added to dilute sulfuric acid, heated in a water bath for 2 hours, the pH was adjusted, centrifuged at 4000 r / min for 15 minutes, the precipitate was washed with distilled water and vacuum dried to obtain pretreated drug residue. The pretreated residue was added to an ethyl acetate-acetone mixed solvent and extracted by ultrasonication; the extracts were combined, concentrated under reduced pressure, and then separated by silica gel column chromatography with petroleum ether-ethyl acetate gradient elution, concentrated and dried to obtain the first powder. The first powder was dissolved in 50% methanol and loaded onto a reversed-phase C18 packed column. The column was eluted with a methanol-water gradient at a flow rate of 2 mL / min. The fraction with a purity >80% was collected, concentrated, and dried to obtain the second powder. The obtained second powder was dissolved in acetone-water mixed solvent by heating, filtered while hot, and the filtrate was allowed to stand at 4°C for 24 hours to crystallize. After filtration, white needle-like crystals were obtained and dried under vacuum to obtain the second hairy cherry extract. The preparation method of the third hairy cherry extract includes: The remaining residue after extracting the second cherry extract was washed with hexane and then vacuum dried. The remaining residue was extracted by reflux with methanol-water, the extracts were combined, concentrated under reduced pressure, and then extracted multiple times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to dryness to obtain the third powder. The third powder was dissolved in methanol and separated by ODS column chromatography with methanol-water gradient elution and HPLC detection. The fractions containing (+)-eugenol were combined, concentrated and dried to obtain the fourth powder. The fourth powder was dissolved in the mobile phase of methanol-0.1% formic acid water and then loaded onto HPLC. The main peak of (+)-eugenol was collected. The collected liquids were combined and concentrated to dryness under reduced pressure to obtain the fifth powder. The fifth powder was dissolved in a methanol-petroleum ether mixture by heating, and allowed to stand at room temperature for 12 hours to precipitate white needle-like crystals. After filtration, the crystals were dried under vacuum at 40°C to obtain the third hairy cherry extract.

3. A film preparation for repairing skin and mucous membrane damage, characterized in that, The membrane formulation comprises, by weight, 10%~25% first-fleshed cherry extract, 1%~3% second-fleshed cherry extract, 0.5%~1.5% third-fleshed cherry extract, 0.1%-1% vitamin B12, 20%~30% chitosan, 10%~20% gelatin, and 0.01%~2% tannic acid; The first hairy cherry extract contains kaempferol and quercetin; the second hairy cherry extract contains 3-p-coumaryl-1,4,6'-triacetylsucrose and 3-p-coumaryl-2',3',6'-triacetylsucrose; and the third hairy cherry extract contains (+)-eugenol. The preparation method of the first hairy cherry extract includes: Take dried Prunus pubescens fruit powder, add 60% ethanol solution and reflux to extract; combine the extracts and concentrate under reduced pressure until there is no alcohol odor to obtain a concentrated solution; pass the concentrated solution through a macroporous adsorption resin column and elute with distilled water, 30% ethanol and 70% ethanol in sequence, collect the 70% ethanol eluent, concentrate under reduced pressure and freeze dry to obtain the first Prunus pubescens extract containing kaempferol and quercetin. The preparation method of the second hairy cherry extract includes: The residue after extracting the first cherry extract was washed with distilled water and dried to constant weight. The dried residue was mixed with distilled water at a solid-liquid ratio of 1:

15. After adjusting the pH, a compound enzyme was added, stirred evenly, and then transferred to a constant temperature shaker for enzymatic hydrolysis. The mixture was heated in an 80°C water bath for 15 minutes, cooled to room temperature, and centrifuged at 4000 r / min for 10 minutes. The precipitate was retained. The compound enzyme was a mixture of cellulase, pectinase, and xylanase. The precipitate after enzymatic hydrolysis was added to dilute sulfuric acid, heated in a water bath for 2 hours, the pH was adjusted, centrifuged at 4000 r / min for 15 minutes, the precipitate was washed with distilled water and vacuum dried to obtain pretreated drug residue. The pretreated residue was added to an ethyl acetate-acetone mixed solvent and extracted by ultrasonication; the extracts were combined, concentrated under reduced pressure, and then separated by silica gel column chromatography with petroleum ether-ethyl acetate gradient elution, concentrated and dried to obtain the first powder. The first powder was dissolved in 50% methanol and loaded onto a reversed-phase C18 packed column. The column was eluted with a methanol-water gradient at a flow rate of 2 mL / min. The fraction with a purity >80% was collected, concentrated, and dried to obtain the second powder. The obtained second powder was dissolved in acetone-water mixed solvent by heating, filtered while hot, and the filtrate was allowed to stand at 4°C for 24 hours to crystallize. After filtration, white needle-like crystals were obtained and dried under vacuum to obtain the second hairy cherry extract. The preparation method of the third hairy cherry extract includes: The remaining residue after extracting the second cherry extract was washed with hexane and then vacuum dried. The remaining residue was extracted by reflux with methanol-water, the extracts were combined, concentrated under reduced pressure, and then extracted multiple times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to dryness to obtain the third powder. The third powder was dissolved in methanol and separated by ODS column chromatography with methanol-water gradient elution and HPLC detection. The fractions containing (+)-eugenol were combined, concentrated and dried to obtain the fourth powder. The fourth powder was dissolved in the mobile phase of methanol-0.1% formic acid water and then loaded onto HPLC. The main peak of (+)-eugenol was collected. The collected liquids were combined and concentrated to dryness under reduced pressure to obtain the fifth powder. The fifth powder was dissolved in a methanol-petroleum ether mixture by heating, and allowed to stand at room temperature for 12 hours to precipitate white needle-like crystals. After filtration, the crystals were dried under vacuum at 40°C to obtain the third hairy cherry extract.

4. The application of *Prunus persica* extract in the preparation of pharmaceutical formulations for repairing skin and mucous membrane damage, characterized in that... The pharmaceutical preparation is at least one of gel, ointment and film preparation; the hairy cherry extract includes a first hairy cherry extract, a second hairy cherry extract and a third hairy cherry extract; The first hairy cherry extract contains kaempferol and quercetin; the second hairy cherry extract contains 3-p-coumaryl-1,4,6'-triacetylsucrose and 3-p-coumaryl-2',3',6'-triacetylsucrose; and the third hairy cherry extract contains (+)-eugenol. The preparation method of the first hairy cherry extract includes: Take dried Prunus pubescens fruit powder, add 60% ethanol solution and reflux to extract; combine the extracts and concentrate under reduced pressure until there is no alcohol odor to obtain a concentrated solution; pass the concentrated solution through a macroporous adsorption resin column and elute with distilled water, 30% ethanol and 70% ethanol in sequence, collect the 70% ethanol eluent, concentrate under reduced pressure and freeze dry to obtain the first Prunus pubescens extract containing kaempferol and quercetin. The preparation method of the second hairy cherry extract includes: The residue after extracting the first cherry extract was washed with distilled water and dried to constant weight. The dried residue was mixed with distilled water at a solid-liquid ratio of 1:

15. After adjusting the pH, a compound enzyme was added, stirred evenly, and then transferred to a constant temperature shaker for enzymatic hydrolysis. The mixture was heated in an 80°C water bath for 15 minutes, cooled to room temperature, and centrifuged at 4000 r / min for 10 minutes. The precipitate was retained. The compound enzyme was a mixture of cellulase, pectinase, and xylanase. The precipitate after enzymatic hydrolysis was added to dilute sulfuric acid, heated in a water bath for 2 hours, the pH was adjusted, centrifuged at 4000 r / min for 15 minutes, the precipitate was washed with distilled water and vacuum dried to obtain pretreated drug residue. The pretreated residue was added to an ethyl acetate-acetone mixed solvent and extracted by ultrasonication; the extracts were combined, concentrated under reduced pressure, and then separated by silica gel column chromatography with petroleum ether-ethyl acetate gradient elution, concentrated and dried to obtain the first powder. The first powder was dissolved in 50% methanol and loaded onto a reversed-phase C18 packed column. The column was eluted with a methanol-water gradient at a flow rate of 2 mL / min. The fraction with a purity >80% was collected, concentrated, and dried to obtain the second powder. The obtained second powder was dissolved in acetone-water mixed solvent by heating, filtered while hot, and the filtrate was allowed to stand at 4°C for 24 hours to crystallize. After filtration, white needle-like crystals were obtained and dried under vacuum to obtain the second hairy cherry extract. The preparation method of the third hairy cherry extract includes: The remaining residue after extracting the second cherry extract was washed with hexane and then vacuum dried. The remaining residue was extracted by reflux with methanol-water, the extracts were combined, concentrated under reduced pressure, and then extracted multiple times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to dryness to obtain the third powder. The third powder was dissolved in methanol and separated by ODS column chromatography with methanol-water gradient elution and HPLC detection. The fractions containing (+)-eugenol were combined, concentrated and dried to obtain the fourth powder. The fourth powder was dissolved in the mobile phase of methanol-0.1% formic acid water and then loaded onto HPLC. The main peak of (+)-eugenol was collected. The collected liquids were combined and concentrated to dryness under reduced pressure to obtain the fifth powder. The fifth powder was dissolved in a methanol-petroleum ether mixture by heating, and allowed to stand at room temperature for 12 hours to precipitate white needle-like crystals. After filtration, the crystals were dried under vacuum at 40°C to obtain the third hairy cherry extract.

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