Medical recombinant collagen repair liquid and preparation method thereof

Abstract

The present application belongs to the technical field of medical biological repair materials, and in particular relates to a medical recombinant collagen repair solution and a preparation method thereof. The present application provides a medical recombinant collagen composition, which comprises: recombinant collagen, glycopeptide and magnolia biondii chenol; wherein the mass ratio of the recombinant collagen, glycopeptide and magnolia biondii chenol is 1:(1-2):(0.3-0.5). The present application scientifically compounds the recombinant collagen, glycopeptide and magnolia biondii chenol, and the three synergistically act to accelerate skin barrier repair from multiple dimensions such as promoting cell proliferation, anti-inflammatory repair and antioxidant. Through the synergy of the glycopeptide and magnolia biondii chenol, a complementary effect is formed with the recombinant collagen from the aspects of double anti-inflammatory pathways and antioxidant. After further adding plant extracts, the repair effect is further enhanced from multiple dimensions such as promoting endogenous collagen synthesis, up-regulating barrier proteins and increasing moisturizing factors.

Description

Technical Field

[0001] This invention belongs to the field of medical biological repair materials technology, specifically, it relates to a medical recombinant collagen repair solution and its preparation method. Background Technology

[0002] Collagen is one of the most abundant proteins in human tissues, widely found in connective tissues such as skin, bones, tendons, and ligaments, playing a vital role in maintaining tissue structure and function. With the development of genetic engineering technology, recombinant collagen has been widely used in medical repair materials, tissue engineering, and medical aesthetics due to its advantages such as good biocompatibility, low immunogenicity, and scalability.

[0003] Currently, various repair products with recombinant collagen as the main active ingredient have been disclosed in existing technologies. For example, patent CN120586023A discloses a medical recombinant collagen repair solution, the raw materials of which include recombinant collagen, sodium hyaluronate, glycerin, purified water, and methylparaben. CN116920164A discloses a repair water containing recombinant type III humanized collagen, which is composed of recombinant type III humanized collagen, hydroxyethyl cellulose, propylene glycol, pentylene glycol, PE9010, and purified water. In addition, CN119868653A discloses a recombinant type III human collagen composition, which includes recombinant type III human collagen, buffer solution, and stabilizer (selected from at least one of glycerin, butylene glycol, pentylene glycol, mannitol, and trehalose).

[0004] However, the existing technology still has the following shortcomings: (1) Most repair solutions have relatively simple formulations and single active ingredients, making it difficult to achieve synergistic repair effects through multiple pathways and targets; (2) Some products use preservatives (such as methylparaben and parabens) which pose certain safety risks and may cause skin irritation or allergic reactions; (3) The stability of existing repair solutions needs to be improved, especially under high temperature or long-term storage conditions, the activity of recombinant collagen is easily reduced; (4) For the repair of skin barrier damage, existing products still have room for improvement in promoting cell migration and reducing inflammatory response.

[0005] Therefore, developing a medical recombinant collagen repair solution that is safe, stable, and has synergistic repair effects has significant clinical application value and market prospects in this field. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a medical recombinant collagen repair solution and its preparation method. Through optimized formulation and preparation process, this invention results in a repair solution with excellent biocompatibility, superior stability, and significant skin repair effects.

[0007] Therefore, the present invention provides the following technical solution.

[0008] The first aspect of the present invention provides a medical recombinant collagen composition comprising: recombinant collagen, glycopeptides and magnolol; wherein the mass ratio of the recombinant collagen, glycopeptides and magnolol is 1:(1~2):(0.3~0.5).

[0009] In a preferred embodiment of the present invention, the recombinant collagen is recombinant type III humanized collagen with a molecular weight of 10~30kDa.

[0010] In this invention, "glycopeptide" refers to polypeptide compounds containing glycan chains, including but not limited to ovomucoid glycopeptide, casein glycopeptide, and chicken egg transferrin glycopeptide. All of the above-mentioned glycopeptides can be obtained from the corresponding natural proteins through enzymatic hydrolysis, separation, and purification using methods known in the art. In a preferred embodiment of the present invention, the glycopeptide is selected from one or a mixture of two or more of ovomucoid glycopeptides, casein glycopeptides, and chicken egg transferrin glycopeptides.

[0011] Casein glycopeptides are a class of polypeptides containing glycans, produced by the degradation of κ-casein by rennet. Studies have shown that casein glycomacropeptides (GMPs) possess anti-inflammatory, antioxidant, and skin barrier support properties. In vitro, in vivo, and limited clinical evidence suggests that GMPs can modulate inflammatory responses, reduce oxidative stress, support collagen integrity, and promote wound healing. In a keratinocyte-mediated atopic dermatitis model, GMPs have been shown to possess anti-inflammatory and antioxidant properties and can stimulate wound closure.

[0012] Egg transferrin is an iron-binding glycoprotein found in egg white. Natural egg transferrin and its derived peptides have been reported to possess antibacterial, antioxidant, anticancer, and immunomodulatory activities. Furthermore, egg transferrin also exhibits anti-inflammatory functions. Egg white proteins (such as lysozyme and egg transferrin), due to their proven antioxidant and antibacterial activities, are being used to enrich advanced wound dressings to help control oxidative stress in wounds, thereby accelerating wound healing and / or preventing bacterial infection.

[0013] Low molecular weight ovomucoid hydrolysate exhibits anti-inflammatory activity in TNF-α-induced human dermal fibroblasts and can inhibit the activation of the NF-κB pathway.

[0014] In a preferred embodiment of the present invention, the magnolol is selected from one or a mixture of two of magnolol A and magnolol B. In the present invention, "magnoliol" refers to a class of lignan compounds, including but not limited to magnolol A and magnolol B. The chemical structure of magnolol A is 12,13-diacetoxy-eucalyptane-4α,6α,11-triol, and the chemical structure of magnolol B is 12,13-diacetoxy-5αH-guaiacol-1(10)-ene-4α,6α,11-triol. Both of the above magnolols can be extracted and isolated from Magnolia species (such as Magnolia officinalis and Magnolia grandiflora) using methods known in the art, or prepared by chemical synthesis.

[0015] Magnoliaol is one of the main active compounds in Magnolia species (such as Magnolia officinalis and Magnolia grandiflora) and possesses significant anti-inflammatory effects. Magnoliaol can significantly inhibit the secretion of pro-inflammatory cytokines. Studies have shown that Magnoliaol can reduce the secretion of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in macrophage inflammatory cells. In a psoriatic dermatitis model, topical application of Magnoliaol improved dermatitis symptoms by inhibiting the NLRP3 / Caspase-1 pathway and regulating tryptophan metabolism. Furthermore, Magnoliaol can also inhibit the release of TRPV3 ion channels (highly expressed in keratinocytes and playing an important role in skin function) and pro-inflammatory cytokines (IL-6 and IL-8) in human epidermal keratinocytes. In addition, Magnoliaol exhibits antioxidant, anti-inflammatory, antibacterial, and healing-promoting activities. Magnoliaol also has anti-glycation effects, effectively improving skin tone and texture, and enhancing skin microcirculation. Lotus and Magnolia leaf extract is classified as a skin conditioning agent and antioxidant.

[0016] This invention combines recombinant collagen, glycopeptides, and magnolol in a specific ratio, enabling them to synergistically promote skin repair. Specifically, glycopeptides exert anti-inflammatory, antioxidant, and repair-promoting effects through multiple mechanisms, including inhibiting the NF-κB inflammatory pathway, scavenging free radicals, and supporting collagen integrity; magnolol reduces the release of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β by inhibiting the NLRP3 / Caspase-1 inflammatory pathway and the TRPV3 channel in keratinocytes; and recombinant collagen provides structural support and signaling guidance for cell migration and tissue regeneration. The three components work synergistically across three dimensions—anti-inflammatory, antioxidant, and collagen protection and regeneration—significantly outperforming the individual use of each component.

[0017] A second aspect of the present invention provides a medical recombinant collagen repair solution, the repair solution comprising the medical recombinant collagen composition as described above.

[0018] In a preferred embodiment of the present invention, the repair solution comprises the following raw material components by mass percentage: 0.5-2% medical recombinant collagen composition, 0.05-0.3% plant extract, 0.01-0.15% sodium hyaluronate, 0.15-0.35% carbomer, and the balance being purified water.

[0019] In this invention, the "plant extract" refers to an extract with skin-repairing activity obtained from plant raw materials through extraction and separation, selected from Centella asiatica (… Centella asiatica ) extract, aloe vera ( Aloe vera ) extract, purslane ( Purslane ) extract, rose ( Rosa rugosa ) extract, citrus ( Citrus The above-mentioned plant extracts are one or more mixtures of fruit peel extracts (spp.). All of the above-mentioned plant extracts can be prepared by conventional extraction methods in the art (such as solvent extraction, ultrasound-assisted extraction, supercritical fluid extraction, etc.).

[0020] In a preferred embodiment of the present invention, the plant extract is selected from one or a mixture of two or more of the following: Centella asiatica extract, aloe vera extract, purslane extract, rose extract, and citrus peel extract.

[0021] Centella asiatica, a plant belonging to the genus Centella in the family Apiaceae, was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and has a long history of application. Its extracts, used in skincare products, primarily contain asiatic acid, asiaticotropic acid, asiaticoside, and asiaticoside. These extracts possess properties that promote skin damage repair, have anti-inflammatory effects, provide antioxidant benefits, and improve the skin barrier.

[0022] Aloe vera extract can promote cell proliferation and migration, and has anti-inflammatory, antibacterial, and immunomodulatory effects. In addition, components in aloe vera extract, such as aloin, have a repairing effect on skin wounds. Aloe vera also has natural moisturizing and water-locking effects, promoting the degradation of fibropolymers into natural moisturizing factors (NMF), increasing the skin's immediate water content, and reducing transepidermal water loss through a mild film-forming effect. Aloe polysaccharides also have a certain soothing effect.

[0023] Purslane extract is rich in polysaccharides, flavonoids, alkaloids, and other compounds with broad-spectrum antibacterial properties, exhibiting good inhibitory effects against common bacteria and fungi. These components also possess anti-inflammatory and anti-allergic effects, reducing inflammation-related cytokines, alleviating or inhibiting skin irritation from common allergens, and weakening skin inflammation. Purslane extract can repair the skin barrier, nourish the skin, and provide moisturizing benefits. Studies have shown that purslane can improve skin itching in mice with allergic contact dermatitis and repair skin barrier function; its mechanism of action may be related to the upregulation of Claudin-1 and Occludin protein expression in skin tissue. Purslane extract can also significantly upregulate the expression of Caspase-14 and Filaggrin genes in the skin barrier system of eczema-affected animals, accelerating the production of natural moisturizing factors and maintaining skin barrier function. Purslane extract contains various phenolic components, possessing certain antioxidant effects, capable of scavenging various free radicals, and inhibiting cellular oxidative damage and aging caused by free radicals. Purslane extract can inhibit the production of reactive oxygen species (ROS) in keratinocytes and increase the production of type I procollagen, thereby exerting an anti-photoaging effect. Purslane extract can also reduce the activity of tyrosinase (a key enzyme in melanin synthesis), thus exerting a whitening effect.

[0024] The polyphenols and flavonoids in roses can significantly scavenge free radicals and upregulate the activity of skin cells' own antioxidant enzymes (such as SOD and GSH-Px), thereby protecting collagen and elastin fibers and delaying the formation of wrinkles and skin sagging. Rose cell sap also has excellent antioxidant activity, inhibiting mitochondrial reactive oxygen species (ROS) levels and increasing the levels of nuclear factor E2-related factor 2 (Nrf2), filaggrin, and nautiloidin. Rose extract can effectively inhibit the release of key inflammatory factors (such as TNF-α and IL-6) and, by regulating classic inflammatory signaling pathways such as NF-κB, fundamentally reduce skin redness, swelling, and inflammatory damage. Rose polysaccharides have good hygroscopic and film-forming properties, effectively locking in moisture and relieving dryness. Rose polysaccharides can activate aquaporin AQP-3, repairing the skin barrier and enhancing moisturizing effects. The active ingredients in roses can improve local blood circulation, ensuring more effective delivery of oxygen and nutrients to skin cells while accelerating the excretion of metabolic waste.

[0025] Citrus peel extracts possess significant antioxidant properties. Noriheterin can exert its antioxidant effect by inhibiting NO production. Citrus peel extracts can inhibit oxidative stress-induced matrix metalloproteinase-1 (MMP-1) expression, exerting its effect by inhibiting the ERK MAPK signaling pathway. Noriheterin can reduce inflammation by decreasing prostaglandin E2 (PGE2) synthesis in keratinocytes. Citrus peel extracts can also inhibit interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α). Citrus peel extracts have a protective effect against skin photoaging. Yuja peel extract has the effects of improving skin wrinkles, moisturizing, and whitening.

[0026] This invention, based on recombinant collagen, glycopeptides, and magnolol, further adds plant extracts. The active ingredients in the plant extracts (such as triterpenoids, flavonoids, polysaccharides, etc.) synergistically exert skin repair effects with the aforementioned components through the following mechanisms: (1) promoting fibroblast proliferation and collagen synthesis (e.g., asiaticoside); (2) inhibiting inflammatory signaling pathways (e.g., the NF-κB pathway) and reducing the release of pro-inflammatory factors (e.g., rose, purslane); (3) scavenging free radicals and protecting the extracellular matrix from oxidative damage (e.g., rose, citrus); (4) upregulating the expression of skin barrier-related proteins (e.g., Claudin-1, Filaggrin, etc.) and maintaining barrier function (e.g., purslane); (5) increasing natural moisturizing factors and improving skin hydration (e.g., aloe vera, rose). Through the above-mentioned synergistic effects of multiple targets and pathways, the skin repair effect of the repair solution of this invention is significantly better than that of each component used alone.

[0027] In a preferred embodiment of the present invention, the sodium hyaluronate has a molecular weight of 10-20 kDa. The sodium hyaluronate can be obtained by extraction from animal tissues (such as rooster combs) using methods known in the art, or by microbial fermentation (such as fermentation using Streptococcus zooepidemicus).

[0028] In a preferred embodiment of the present invention, the carbomer is any one of carbomer 940, carbomer 941 or carbomer 934.

[0029] A third aspect of the present invention provides a method for preparing the medical recombinant collagen repair solution as described above, comprising the following steps: (1) Heat purified water to 65~75℃, add carbomer and stir until completely dissolved to obtain an aqueous mixture; (2) At 20~30℃, add the recombinant collagen composition, plant extract and sodium hyaluronate to the obtained aqueous mixture in sequence, and stir until completely dissolved to obtain a mixture; (3) The obtained mixture is filtered through a 0.22μm filter membrane for sterilization and then filled into a sterile container under sterile conditions to obtain medical recombinant collagen repair solution.

[0030] By employing the above technical solution, the present invention has at least the following advantages: (1) Significant Synergistic Repair Efficacy: This invention scientifically combines recombinant collagen with glycopeptides and magnolia extract. The three work synergistically to accelerate skin barrier repair from multiple dimensions, including promoting cell proliferation, anti-inflammatory repair, and anti-oxidation. Glycopeptides (ovomucoid glycopeptide, casein glycopeptide, chicken egg transferrin glycopeptide) exert anti-inflammatory, antioxidant, and repair-promoting effects through multiple mechanisms, such as inhibiting the NF-κB inflammatory pathway, scavenging free radicals, and supporting collagen integrity. Magnolia extract reduces the release of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β by inhibiting the NLRP3 / Caspase-1 inflammatory pathway and the TRPV3 channel of keratinocytes. The two work synergistically to complement and enhance the effect of recombinant collagen from the perspectives of dual anti-inflammatory pathways and anti-oxidation. The addition of plant extracts further enhances the repair effect from multiple dimensions, such as promoting endogenous collagen synthesis, upregulating barrier proteins, and increasing moisturizing factors.

[0031] (2) High safety of the formula: This invention does not add traditional parabens, thus avoiding the risk of skin irritation and allergies, and has good safety.

[0032] (3) Excellent product stability: The present invention can effectively protect the activity of recombinant collagen during storage by combining glycopeptides with recombinant collagen, prevent protein denaturation and aggregation, and extend the shelf life of the product.

[0033] (4) Good biocompatibility: All raw materials of this invention are medical grade or pharmaceutical grade excipients, without any chemical cross-linking agents or enzyme cross-linking agents, and are non-cytotoxic, non-sensitizing, and have good biocompatibility and degradability.

[0034] (5) The preparation process is simple and controllable: The preparation method of the present invention is simple and mild, and does not involve severe conditions such as high temperature and high pressure. It can maintain the biological activity of recombinant collagen to the maximum extent and is suitable for industrial production.

[0035] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Detailed Implementation

[0036] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0037] Unless otherwise specified, the preparation of ovomucoid glycopeptides involved in the embodiments of the present invention can refer to the content disclosed in patent CN121622864A, specifically including: taking egg white from fresh eggs, adding 0.1 mol / L NaCl solution at a volume ratio of 1:1, and stirring at 4 ℃ for 1 h; adjusting the pH of the diluted egg white solution to 6.5, adding 3% polyethylene glycol 8000, and stirring thoroughly at 4 ℃ for 2 h; centrifuging the egg white mixture (4 ℃, 15000×g, 15 min) to remove the supernatant, and collecting the precipitate as crude ovomucoid; then, adding 0.5 mol / L NaCl solution to the crude ovomucoid at a ratio of 1:2 (g:g), treating at 4 ℃ and 300 rpm for 4 h, centrifuging (15000×g, 15 min) to collect the precipitate, and repeating twice; then washing twice with ultrapure water at a ratio of 1:2 (g:g) under the same conditions, and collecting the precipitate as purified ovomucoid. A certain amount of ovomucoid was weighed into a beaker and dissolved in PBS (1×). The protein solution was adjusted to pH 2.0 with 1 mol / L hydrochloric acid, and pepsin (1:25 U / mg) was added. The beaker was then placed in a preheated 37°C shaking incubator for 2 h of digestion. The pH of the digestion solution was then adjusted to 7.8, and trypsin (1:25 U / mg) and α-chymotrypsin (1:100 U / mg) were added. The beaker was then placed in a 37°C shaking incubator for another 6 h of digestion to obtain the ovomucoid polypeptide digest. The digestion solution was filtered through a 0.25 μm microporous membrane, and the filtrate was collected. Con A Sepharose TMThe 4B affinity chromatography column was pre-swollen for 30 min in 0.1 M acetate buffer (pH 6.0) containing 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2. The column was then washed with 10 column volumes of 20 mM Tris-HCl binding buffer (pH 7.4). After injecting 2 column volumes of the filtrate, the column was washed with 10 column volumes of binding buffer, followed by elution with binding buffer containing 0.2 M and 0.5 M α-D-methylmannoside, collecting 4 column volumes of 0.2 M eluent. Following elution, the column was washed with 5 column volumes of binding buffer, and the column was stored in binding buffer containing 20% ​​ethanol. The 0.2 M eluent was then placed in a 500-1000 D dialysis bag for dialyzing at 4°C for 36 h at a sample-to-dialysis buffer ratio of 1:200, with three buffer changes. After dialysis of the eluent, it was concentrated to dryness under vacuum to obtain ovomucin glycopeptide dry powder.

[0038] Unless otherwise specified, the preparation of chicken egg transferrin glycopeptides involved in the embodiments of this invention can refer to the content disclosed in patent CN116041485A, specifically including: dissolving chicken egg transferrin in PBS (3X) (1:2 w / w), adding two equal volumes of deionized water to the resulting mixture, adjusting the pH to 2.0 with 1M HCl, adding pepsin (1:25 w / w), and incubating at 37°C for 2 hours with stirring every 30 minutes. Adjusting the pH of the hydrolysate to 7.8 with 1M NaOH, adding trypsin (1:25 w / w) and chymotrypsin α-Chymotrypsin (1:100 w / w), and incubating at 37°C for 6 hours with stirring every 30 minutes. Filtering the resulting digest through a 0.25 μm microporous membrane and collecting the filtrate. Con A Sepharose TMThe 4B affinity chromatography column was pre-swollen for 30 min in 0.1 M acetate buffer (pH 6.0) containing 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2. The column was then washed with 10 column volumes of 20 mM Tris-HCl binding buffer (pH 7.4). After injecting 2 column volumes of the filtrate, the column was washed with 10 column volumes of binding buffer, followed by elution with binding buffer containing 0.2 M and 0.5 M α-D-methylmannoside, collecting 4 column volumes of 0.2 M eluent. Following elution, the column was washed with 5 column volumes of binding buffer, and the column was stored in binding buffer containing 20% ​​ethanol. The 0.2 M eluent was then placed in a 500-1000 D dialysis bag for dialyzing at 4°C for 36 h, with a sample-to-dialysis buffer ratio of 1:200, and the buffer was changed 3 times. After dialysis of the eluent, the solution was concentrated to dryness under vacuum to obtain chicken egg transferrin glycopeptide powder. The preparation method of casein glycopeptide is the same as that of chicken egg transferrin glycopeptide, except that the raw material chicken egg transferrin is replaced with casein, which will not be described in detail here.

[0039] Unless otherwise specified, the preparation process of the plant extracts involved in the following examples is as follows: The plant raw materials to be extracted (whole Centella asiatica, aloe vera leaves, whole Portulaca oleracea, rose petals, and citrus peel) are dried and pulverized separately, and then passed through a 40-mesh sieve. The plant powder is weighed and 10 times (volume / mass) of 95% ethanol solution is added. After soaking at room temperature for 36 hours, the mixture is heated and refluxed three times, with each extraction lasting 2 hours at an extraction temperature of 70-80°C. The three extracts are combined, filtered, and the filtrate is subjected to reduced pressure at 50°C to recover the ethanol until no alcohol odor remains. The extract is then concentrated to a relative density of 1.10-1.15 (60°C). The extract is dissolved in an appropriate amount of purified water and extracted three times with an equal volume of chloroform until the extract is colorless. The chloroform extracts are combined, dried over anhydrous sodium sulfate, and the solvent is recovered under reduced pressure at 40°C. The extract is then freeze-dried (-50°C, vacuum <10Pa, 24 hours), pulverized, and the plant extract powder is obtained. The extraction conditions are the same for each plant extract; only the raw materials differ.

[0040] Example 1: Medical Recombinant Collagen Repair Solution I and its Preparation The medical recombinant collagen composition of this embodiment is composed of recombinant type III humanized collagen (molecular weight 20kDa), ovomucoid glycopeptide and magnolol A in a mass ratio of 1:1.5:0.4.

[0041] The medical recombinant collagen repair solution I of this embodiment is composed of the following raw materials in weight percentage: 1.0% medical recombinant collagen composition, 0.15% plant extract (a mixture of Centella asiatica extract, aloe vera extract, and purslane extract in a weight ratio of 1:2:1), 0.08% sodium hyaluronate (molecular weight 15kDa), 0.25% carbomer 940, and the balance being purified water.

[0042] The preparation steps of the medical recombinant collagen repair solution I in this embodiment include: (1) heating purified water to 70°C, adding carbomer 940 and stirring until completely dissolved to obtain an aqueous phase mixture. (2) adding recombinant collagen composition, plant extract and sodium hyaluronate sequentially to the obtained aqueous phase mixture at 25°C, stirring until completely dissolved to obtain a mixture. (3) filtering the obtained mixture through a 0.22μm filter membrane for sterilization, and filling it into a sterile container under aseptic conditions to obtain medical recombinant collagen repair solution I.

[0043] Example 2: Medical Recombinant Collagen Repair Solution II and its Preparation The medical recombinant collagen composition of this embodiment is composed of recombinant type III humanized collagen (molecular weight 10kDa), casein glycopeptide and magnolol B in a mass ratio of 1:1:0.3.

[0044] The medical recombinant collagen repair solution II of this embodiment is composed of the following raw materials in weight percentage: 0.5% medical recombinant collagen composition, 0.05% plant extract (a mixture of Centella asiatica extract and Portulaca oleracea extract in a 1:1 mass ratio), 0.01% sodium hyaluronate (molecular weight 10kDa), 0.15% carbomer 941, and the balance being purified water.

[0045] The preparation steps of the medical recombinant collagen repair solution II in this embodiment include: (1) heating purified water to 65°C, adding carbomer 941 and stirring until completely dissolved to obtain an aqueous phase mixture. (2) adding the recombinant collagen composition, plant extract and sodium hyaluronate sequentially to the obtained aqueous phase mixture at 30°C, stirring until completely dissolved to obtain a mixture. (3) filtering the obtained mixture through a 0.22μm filter membrane for sterilization, and filling it into a sterile container under aseptic conditions to obtain the medical recombinant collagen repair solution II.

[0046] Example 3: Medical Recombinant Collagen Repair Solution III and its Preparation The medical recombinant collagen composition III of this embodiment is composed of recombinant type III humanized collagen (molecular weight 30kDa), chicken egg transferrin glycopeptide and magnolol A in a mass ratio of 1:2:0.5.

[0047] The medical recombinant collagen repair solution III of this embodiment is composed of the following raw materials in weight percentage: 2.0% medical recombinant collagen composition, 0.3% plant extract (a mixture of Centella asiatica extract and Aloe vera extract in a weight ratio of 1:2), 0.15% sodium hyaluronate (molecular weight 20kDa), 0.35% carbomer 934, and the balance being purified water.

[0048] The preparation steps of the medical recombinant collagen repair solution III in this embodiment include: (1) heating purified water to 75°C, adding carbomer 940 and stirring until completely dissolved to obtain an aqueous phase mixture. (2) adding the recombinant collagen composition, plant extract and sodium hyaluronate sequentially to the obtained aqueous phase mixture at 20°C, stirring until completely dissolved to obtain a mixture. (3) filtering the obtained mixture through a 0.22μm filter membrane for sterilization, and filling it into a sterile container under aseptic conditions to obtain the medical recombinant collagen repair solution III.

[0049] Example 4: Medical Recombinant Collagen Repair Solution IV and its Preparation The medical recombinant collagen composition of this embodiment is composed of recombinant type III humanized collagen (molecular weight 20kDa), ovomucoid glycopeptide, casein glycopeptide and magnolol B in a mass ratio of 1:1:1:0.3.

[0050] The medical recombinant collagen repair solution IV of this embodiment is composed of the following raw materials in weight percentage: 1% medical recombinant collagen composition, 0.2% plant extract (a mixture of aloe vera extract and purslane extract in a 1:1 weight ratio), 0.08% sodium hyaluronate (molecular weight 15kDa), 0.20% carbomer 940, and the balance being purified water.

[0051] The preparation steps of the medical recombinant collagen repair solution IV in this embodiment include: (1) heating purified water to 70°C, adding carbomer 940 and stirring until completely dissolved to obtain an aqueous phase mixture. (2) adding the recombinant collagen composition, plant extract and sodium hyaluronate sequentially to the obtained aqueous phase mixture at 25°C, stirring until completely dissolved to obtain a mixture. (3) filtering the obtained mixture through a 0.22μm filter membrane for sterilization, and filling it into a sterile container under aseptic conditions to obtain the medical recombinant collagen repair solution IV.

[0052] Example 5: Medical Recombinant Collagen Repair Solution V and its Preparation The medical recombinant collagen composition of this embodiment is composed of recombinant type III humanized collagen (molecular weight 10kDa), casein glycopeptide, chicken egg transferrin glycopeptide and magnolol A in a mass ratio of 1:0.5:0.5:0.5.

[0053] The medical recombinant collagen repair solution V of this embodiment is composed of the following raw materials in the following mass percentages: 1.5% medical recombinant collagen composition, 0.3% plant extract (a mixture of purslane extract, rose extract, and citrus peel extract in a mass ratio of 1:1:2), 0.12% sodium hyaluronate (molecular weight 20kDa), 0.35% carbomer 940, and the balance being purified water.

[0054] The preparation steps of the medical recombinant collagen repair solution V in this embodiment include: (1) heating purified water to 75°C, adding carbomer 940 and stirring until completely dissolved to obtain an aqueous phase mixture. (2) adding recombinant collagen composition, plant extract and sodium hyaluronate sequentially to the obtained aqueous phase mixture at 25°C, stirring until completely dissolved to obtain a mixture. (3) filtering the obtained mixture through a 0.22μm filter membrane for sterilization, and filling it into a sterile container under aseptic conditions to obtain the medical recombinant collagen repair solution V.

[0055] Comparative Example 1: Medical Recombinant Collagen Repair Solution A and its Preparation The only difference between this comparative example and Example 1 is that the medical recombinant collagen composition is composed of recombinant type III humanized collagen (molecular weight 20kDa), ovomucoid glycopeptide and magnolia alcohol A in a mass ratio of 1:0.5:0.1. The remaining components and preparation methods are the same as in Example 1, resulting in medical recombinant collagen repair solution A.

[0056] Comparative Example 2: Medical Recombinant Collagen Repair Solution B and its Preparation The only difference between this comparative example and Example 1 is that the medical recombinant collagen composition is composed of recombinant type III humanized collagen (molecular weight 20kDa), ovomucoid glycopeptide and magnolia alcohol A in a mass ratio of 1:3:0.2. The remaining components and preparation methods are the same as in Example 1, resulting in medical recombinant collagen repair solution B.

[0057] Comparative Example 3: Medical Recombinant Collagen Repair Solution C and its Preparation The only difference between this comparative example and Example 1 is that the medical recombinant collagen composition is composed of recombinant type III humanized collagen (molecular weight 20kDa), ovomucoid glycopeptide and magnolia alcohol A in a mass ratio of 1:3:0.6. The remaining components and preparation methods are the same as in Example 1, resulting in medical recombinant collagen repair solution C.

[0058] Comparative Example 4: Medical Recombinant Collagen Repair Solution D and its Preparation The only difference between this comparative example and Example 1 is that the medical recombinant collagen composition is composed of recombinant type III humanized collagen (molecular weight 20kDa) and ovomucoid glycopeptide in a mass ratio of 1:1.5. The other components and preparation methods are the same as in Example 1, resulting in medical recombinant collagen repair solution D.

[0059] Comparative Example 5: Medical Recombinant Collagen Repair Solution E and its Preparation The only difference between this comparative example and Example 1 is that the medical recombinant collagen composition is composed of recombinant type III humanized collagen (molecular weight 20kDa) and magnolia alcohol A in a mass ratio of 1:0.4. The other components and preparation methods are the same as in Example 1, resulting in medical recombinant collagen repair solution E.

[0060] Comparative Example 6: Medical Recombinant Collagen Repair Solution F and its Preparation The only difference between this comparative example and Example 1 is that the medical recombinant collagen composition is composed of recombinant type III humanized collagen (molecular weight 20kDa), and the other components and preparation methods are the same as in Example 1, resulting in medical recombinant collagen repair solution F.

[0061] Experimental Example 1: In vitro cell proliferation activity experiment This experiment investigated the effects of different repair solutions on the in vitro proliferation activity of human skin fibroblasts, specifically including the following steps: Human skin fibroblasts (HSF) were harvested and seeded into 96-well plates (1 × 10⁶ cells per well). 4 Cells were added to each well with the retrieval solutions (pre-diluted 10-fold with DMEM medium) of Examples 1-5 and Comparative Examples 1-6, respectively. DMEM medium without retrieval solution served as a blank control, and medium containing 0.1% SDS served as a positive control (cytotoxicity control). Each group was divided into 6 replicates. After incubation at 37°C and 5% CO2 for 48 h, 10 μL of CCK-8 reagent was added to each well, and incubation continued for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0062] Cell viability (%) = (OD value of experimental group - OD value of blank control group) / OD value of blank control group × 100% The experimental results are shown in Table 1.

[0063] Table 1. Effects of each group of repair solutions on the proliferation activity of human skin fibroblasts (CCK-8 assay, mean ± SD, n = 6) ; Note: Compared with the blank control, * P<0.05, ** P<0.01; compared with Example 1, # P<0.05 As shown in Table 1, the repair solutions of Examples 1-5 of this invention can significantly promote the proliferation of human skin fibroblasts (P<0.01), with cell survival rates all exceeding 161%. Among them, Example 1 showed the best effect, with a cell survival rate of 175.8%. The proliferation-promoting effects of Comparative Example 4 (without magnolol, cell survival rate 142.5%) and Comparative Example 5 (without glycopeptides, cell survival rate 129.8%) were significantly lower than those of the examples (P<0.05). The proliferation-promoting effect of Comparative Example 6 (without both magnolol and glycopeptides, cell survival rate 97.2%) was not significantly different from the blank control (P>0.05), indicating that magnolol and glycopeptides have a significant synergistic effect with recombinant collagen in promoting cell proliferation, and all three are indispensable. The proliferation-promoting effects of Comparative Examples 1-3 (with ratios outside the scope of this invention) were significantly lower than those of the Examples (P<0.05), indicating that the ratio range of the three (1:1~2:0.3~0.5) plays a key role in the synergistic effect.

[0064] Experimental Example 2: Evaluation of Skin Barrier Repair Effect A mouse dorsal skin barrier damage model was established following the methods reported in the literature. Eighty-four SPF-grade Balb / c mice (half male and half female, weighing 18-22g) were randomly divided into 14 groups: normal control group, model control group, Examples 1-5, and Comparative Examples 1-6, with six mice in each group. Twenty-four hours before the experiment, the mice's dorsal hair (approximately 3cm × 4cm area) was removed with depilatory cream. The normal control group received no treatment. In the other groups, medical tape was repeatedly applied (approximately 20 times) until the transepidermal water loss (TEWL) value increased more than three times compared to normal skin, thus establishing the skin barrier damage model.

[0065] After successful modeling, mice in Examples 1-5 and Comparative Examples 1-6 were treated with the corresponding repair solution (0.2 mL each time) twice daily (8 hours apart) for 7 consecutive days. The model control group was treated with an equal volume of physiological saline, while the normal control group received no treatment. The TEWL values ​​of the dorsal skin of each group of mice were measured using a TEWL analyzer before modeling, immediately after modeling, and before medication on days 1, 3, 5, and 7, and the TEWL recovery rate was calculated.

[0066] TEWL recovery rate (%) = (TEWL value of normal control group - TEWL value of experimental group) / (TEWL value of normal control group - TEWL value of model control group) × 100%.

[0067] The experimental results are shown in Table 2.

[0068] Table 2. Effects of each repair solution on the TEWL recovery rate in mice with skin barrier damage (mean±SD, n=6) ; Note: Compared with the model control group.* P<0.05, ** P<0.01; compared with Example 1, # P<0.05.

[0069] Table 2 shows that the repair solutions of Examples 1-5 all had significant repair effects on skin barrier damage (P<0.01), with TEWL recovery rates all exceeding 90%. Among them, Example 1 showed the best effect (95.2%), approaching the level of the normal control group. The TEWL recovery rates of Comparative Example 4 (without magnolol, TEWL recovery rate 79.6%) and Comparative Example 5 (without glycopeptides, TEWL recovery rate 72.3%) were significantly lower than those of the examples (P<0.05); Comparative Example 6 (without both magnolol and glycopeptides, TEWL recovery rate 53.8%) was significantly lower than those of the examples and Comparative Examples 4 and 5 (P<0.05). These results indicate that magnolol, glycopeptides, and recombinant collagen have a synergistic effect in repairing the skin barrier, and the combined effect of all three is significantly better than any combination of two components. The TEWL recovery rates of Comparative Examples 1-3 (with ratios outside the scope of this invention) were all below 85%, significantly inferior to those of the embodiments (P<0.05), further confirming that the mass ratio range (1:1~2:0.3~0.5) described in this invention is crucial for achieving the best synergistic repair effect.

[0070] Experimental Example 3: Stability Test The repair solutions from Examples 1-5 and Comparative Examples 1-6 were stored in the dark at 4℃, 25℃, and 40℃, respectively, and samples were taken at 0, 1, 3, and 6 months. The activity content of recombinant collagen in each group of repair solutions was determined by a double-antibody sandwich ELISA method. The activity content at 0 months was taken as 100%, and the activity retention rate at each time point was calculated. Each sample was tested in triplicate, and the average value was taken. The results are shown in Tables 3 and 4.

[0071] Table 3. Activity retention rate of each group of remedy solutions after 6 months of storage at 40℃ (ELISA method, mean±SD, n=3) ; Note: Compared with Example 1, **P<0.01 Table 4. Activity retention rate of the remedy solution in Example 1 after storage at different temperatures for different times (ELISA method, mean ± SD, n=3) ; As shown in Table 3, after 6 months of storage at 40°C, the recombinant collagen activity retention rate of Example 1 still reached 94.5%, significantly higher than all comparative examples (P<0.01). The activity retention rates of Comparative Example 4 (without magnolol) and Comparative Example 5 (without glycopeptides) after 6 months of storage at 40°C were 79.8% and 71.5%, respectively, both significantly lower than Example 1 (P<0.01). The activity retention rate of Comparative Example 6 (without both magnolol and glycopeptides) was only 60.3%, the lowest among all groups, showing a highly significant difference compared to Example 1 (P<0.01). These results indicate that the addition of magnolol and glycopeptides can significantly improve the thermal stability of the repair solution, and their synergistic effect can more effectively protect the bioactivity of recombinant collagen.

[0072] As shown in Table 4, the activity retention rates of Example 1 reached 97.8% and 95.8% after 6 months of storage at 4℃ and 25℃, respectively, indicating that the repair solution of the present invention has excellent stability under normal storage conditions.

[0073] Test Example 4: Skin Irritation Test Following the standard method of "Biological Evaluation of Medical Devices Part 10: Skin Irritation and Delayed-Type Hypersensitivity Tests" (GB / T16886.10-2017), skin irritation tests were conducted using healthy New Zealand white rabbits. Forty-two healthy rabbits (half male and half female, weighing 2.0–2.5 kg) were randomly divided into 14 groups: Examples 1–5, Comparative Examples 1–6, a negative control group (physiological saline), and a positive control group (5% SDS solution), with three rabbits in each group.

[0074] Twenty-four hours prior to the experiment, the fur on both sides of the spine on the back of rabbits was removed using an electric shaver (approximately 3cm x 3cm on each side) to ensure the skin remained intact. A self-comparison method was used, with the left side as the treatment side and the right side as the control side. 0.5mL of each repair solution (5% SDS solution for the positive control group and physiological saline for the negative control group) was applied to four layers of gauze measuring 2.5cm x 2.5cm and applied to the skin of the treatment side, secured with non-irritating adhesive tape; an equal volume of physiological saline gauze was applied to the control side. After 4 hours, the dressings were removed, and any residue was washed away with warm water. Skin reactions were observed and recorded at 1h, 24h, 48h, and 72h after dressing removal. Erythema, eschar formation, and edema formation were scored according to standard scoring criteria, and the Primary Irritation Index (PII) was calculated.

[0075] PII = (Sum of stimulus response scores on the administration side at each observation time point - Sum of stimulus response scores on the control side) / Number of observations.

[0076] The experimental results are shown in Table 5.

[0077] Table 5 Results of skin irritation test of repairing solutions in each group on rabbit skin (primary irritation index PII, n=3) ; Note: Judged according to GB / T 16886.10-2017 standard: PII=0 means no irritation, 0<PII≤2 means slight irritation, 2<PII≤5 means moderate irritation, and PII>5 means strong irritation.

[0078] It can be seen from Table 5 that the repairing solutions of Examples 1 to 5 did not cause any skin irritation reactions such as erythema and edema during the whole experiment period (72h), all PII were 0, and were judged as no irritation. No skin irritation reaction was also observed in the repairing solutions of Comparative Examples 1 to 6, all PII were 0, and were judged as no irritation. The above results show that the repairing solution of the present invention has no irritation to skin, has good safety, and the addition and proportion change of Magnolia grandiflora alcohol and glycopeptide do not affect the basic safety of the product.

[0079] Test Example 5: Comparative analysis of comprehensive performance The main performance indicators of each group in the above Test Examples 1 to 4 were comprehensively compared, with each indicator of Example 1 as the benchmark (standardized to 100%), and the comprehensive performance score of each group was calculated. Comprehensive performance score = (relative cell survival rate × 0.30) + (relative TEWL recovery rate × 0.35) + (relative activity retention rate × 0.25) + (safety score × 0.10), wherein, for safety score: 100 points are recorded when PII=0, and points are deducted proportionally when PII>0.

[0080] The experimental results are shown in Table 6.

[0081] Table 6 Comparison of comprehensive performance scores of repairing solutions in each group ; It can be seen from Table 6 that the comprehensive comparative analysis shows that Example 1 has the best comprehensive performance (comprehensive score 100.0), followed by Examples 2 to 5 (comprehensive score 93.1 to 97.6), all of which are significantly better than all comparative examples. Comparative Example 6 (without adding both Magnolia grandiflora alcohol and glycopeptide) has the lowest comprehensive score (53.7), which is only 53.7% of that of Example 1, further confirming the indispensability of the synergistic effect of recombinant collagen, glycopeptide and Magnolia grandiflora alcohol in the present invention. The comprehensive scores of Comparative Examples 1 to 3 (the ratio exceeds the scope of the present invention) are all lower than 79, which are significantly inferior to all examples, indicating that the mass ratio range (1:1~2:0.3~0.5) described in the present invention is a key technical feature to achieve excellent comprehensive performance.

[0082] In summary, this invention, by compounding recombinant collagen, glycopeptides, and magnolol in a specific ratio (1:1~2:0.3~0.5), and supplementing with plant extracts, sodium hyaluronate, and other components, produces a medical recombinant collagen repair solution that exhibits excellent performance in promoting fibroblast proliferation, repairing skin barrier function, and maintaining product stability. Furthermore, it is non-irritating to the skin and has promising clinical application prospects.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A medical recombinant collagen composition, characterized in that, The composition comprises: recombinant collagen, glycopeptides, and magnolol; wherein the mass ratio of the recombinant collagen, glycopeptides, and magnolol is 1:(1~2):(0.3~0.5).

2. The medical recombinant collagen composition according to claim 1, characterized in that, The recombinant collagen is recombinant type III humanized collagen with a molecular weight of 10~30kDa.

3. The medical recombinant collagen composition according to claim 1, characterized in that, The glycopeptide is selected from one or a mixture of two or more of ovomucoid glycopeptides, casein glycopeptides, and chicken egg transferrin glycopeptides.

4. The medical recombinant collagen composition according to claim 1, characterized in that, The magnolia alcohol is selected from one or a mixture of two of magnolia alcohol A and magnolia alcohol B.

5. A medical recombinant collagen repair solution, characterized in that, The repair solution comprises the medical recombinant collagen composition according to any one of claims 1 to 4.

6. The medical recombinant collagen repair solution according to claim 5, characterized in that, The repair solution comprises the following raw material components by mass percentage: 0.5-2% medical recombinant collagen composition, 0.05-0.3% plant extract, 0.01-0.15% sodium hyaluronate, 0.15-0.35% carbomer, and the balance being purified water.

7. The medical recombinant collagen repair solution according to claim 6, characterized in that, The plant extract is selected from one or a mixture of two or more of the following: Centella asiatica extract, aloe vera extract, purslane extract, rose extract, and citrus peel extract.

8. The medical recombinant collagen repair solution according to claim 6, characterized in that, The molecular weight of the sodium hyaluronate is 10~20 kDa.

9. The medical recombinant collagen repair solution according to claim 6, characterized in that, The carbomer is any one of carbomer 940, carbomer 941 or carbomer 934.

10. A method for preparing a medical recombinant collagen repair solution as described in any one of claims 1 to 9, characterized in that, Includes the following steps: (1) Heat purified water to 65~75℃, add carbomer and stir until completely dissolved to obtain an aqueous mixture; (2) At 20~30℃, add the recombinant collagen composition, plant extract and sodium hyaluronate to the obtained aqueous mixture in sequence, and stir until completely dissolved to obtain a mixture; (3) The obtained mixture is filtered through a 0.22μm filter membrane for sterilization and then filled into a sterile container under sterile conditions to obtain medical recombinant collagen repair solution.