A skin extract of axolotl, its preparation method and application

Abstract

The present invention discloses an axolotl skin extract, its preparation method and applications, relating to the technical field of biomedicine. The axolotl skin extract is rich in various active ingredients, including members of the collagen family, fibronectin, C1Q complement protein, fibronectin, etc. These ingredients complement and promote each other, making the extract have remarkable effects in aspects such as skin anti-aging and skin injury repair, and thus having broad application prospects in drug R & D, cosmetics and functional foods. The method for extracting axolotl skin protein, through rapid freeze-thaw treatment of samples, combined with mechanical crushing, ultrasonic-assisted separation and multiple filtration steps, avoids the influence on the activity of the product, and can obtain a variety of composite proteins; adjusting the pH value of the buffer solution further optimizes the properties of the extract, making it more suitable for human skin, and being sterile and directly usable as the raw material of the product, realizing large-scale production and promoting the industrial application of axolotl skin extract.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an axolotl skin extract, a preparation method thereof, and an application thereof. Background Art

[0002] With the continuous progress of biotechnology and natural resource development technologies, the extraction and utilization of natural active ingredients have become a research hotspot in multiple fields such as medicine, cosmetics, and functional foods. Among them, active extracts derived from organisms have attracted much attention due to their high safety, strong biological activity, and multifunctionality. As an important source of natural active ingredients, amphibians are rich in a variety of substances with significant biological activities in their skin secretions and tissues, such as small molecule peptides (such as antioxidant peptides, wound healing promoting peptides, immunomodulatory peptides, antibacterial peptides, antiviral peptides, antitumor peptides, etc.), antioxidant factors, and polysaccharide compounds. These substances show great potential in aspects such as anti-infection, anti-inflammation, and promoting tissue regeneration.

[0003] Among amphibians, caudate species such as salamanders and axolotls have received extensive attention due to their excellent regenerative ability and rich skin secretion characteristics. Research shows that the skin of caudate species contains a variety of active ingredients with antibacterial, antioxidant, antitumor, and immunomodulatory functions, which makes them have great application prospects in the development of medicine and biological products. The antibacterial peptides secreted by the skin of caudate species not only have broad-spectrum antibacterial properties but also can significantly inhibit a variety of drug-resistant strains, providing a potential solution to the problem of antibiotic abuse. In addition, the regeneration factors in the skin of caudate species have important value in promoting cell division and tissue repair and are ideal candidate materials for tissue engineering and regenerative medicine research. However, compared with other amphibians such as frogs and toads, the research on the skin active ingredients of caudate species such as salamanders and axolotls started relatively late, the exploration of their applications is even in its infancy, and the application scope of their skin extracts is also greatly limited.

[0004] Therefore, solving the problems of less research on the skin extracts of current caudate species such as salamanders and axolotls, limited application scope, and serious shortage of application fields, exploring efficient, stable, and sustainable active ingredient extraction technologies, providing a wider range of application fields, and filling the gap in the practical application of amphibian caudate species skin extracts are of great significance for promoting the development of this field. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides an axolotl skin extract, a preparation method thereof, and an application thereof, aiming to solve the problems of less research on the skin extracts of current amphibian caudate species, limited application scope, and serious shortage of application fields.

[0006] An embodiment of the first aspect of the present invention provides a salamander skin extract, and the active ingredients of the salamander skin extract include one or more of the collagen (COL) family, one or more of the ficolin (FCN) family, one or more of the C1Q complement protein family, fibronectin (FN1), cytochrome b5 reductase 3 (CYB5R3), nicotinamide phosphoribosyltransferase (NAMPT), or mitochondrial aldehyde dehydrogenase 2 (ALDH2).

[0007] The salamander skin extract according to the embodiment of the first aspect of the present invention has at least the following beneficial effects: The salamander skin extract provided by the present invention is rich in various active ingredients. Among them, the collagen (Collagen, COL) family and the ficolin (Ficolin, FCN) family can improve skin water content and reduce transdermal water loss of the skin, improve skin wrinkles and elasticity, and can also improve the immune proteins of the skin collagen fiber structure complement system; C1Q complement protein (Complement Component 1q) plays a key role in neuronal function and aging; fibronectin (Fibronectin, FN1 or FN) has the function of a growth factor, can promote cell proliferation, induce epidermal cells to form granulation tissue, and promote the reconstruction of the subepidermal basement membrane and the normal keratinization process. As an important component of the skin extracellular matrix, it can firmly anchor collagen on skin cells, strengthen the elastic fiber network of the skin, tighten the loose and sagging skin, promote the growth, differentiation, and migration of skin cells, guide the orderly regeneration of the skin, promote the expression of barrier proteins, repair the skin barrier, and enhance skin defense; overexpression of cytochrome b5 reductase 3 (cytochrome b5 reductase 3, CYB5R3) activates respiratory metabolism and can play a role in extending lifespan; while cytochrome b5 reductase 3, nicotinamide phosphoribosyltransferase (nicotinamide phosphoribosyltransferase, NAMPT), and mitochondrial aldehyde dehydrogenase 2 (aldehyde dehydrogenase 2, ALDH2) have a promoting effect on metabolism and play an important role in aspects such as cell aging and immune cell function regulation. The above-mentioned rich bioactive ingredients complement and promote each other, making the salamander skin extract have broad application prospects in drug research and development, cosmetics, and functional foods.

[0008] In some embodiments of the present invention, the collagen family includes COL1A1, COL1A2, COL3A1, COL4A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL6A6, COL11A1, COL12A1, COL17A1, COL18A1, COL28A1, or COLGALT1. The Collagen family is the most abundant protein in mammals. The axolotl skin extract of the present invention contains abundant collagen and covers multiple members of its family.

[0009] In some embodiments of the present invention, the ficolin family includes FCN1 (Ficolin-1), FCN2 (Ficolin-2), or FCN3 (Ficolin-3).

[0010] In some embodiments of the present invention, the C1Q complement protein family includes C1QC (Complement C1q Subcomponent C), C1QB (Complement C1q Subcomponent B), C1QA (Complement C1q Subcomponent A), or C1QTNF3 (Clq and TNF related 3).

[0011] In some embodiments of the present invention, the axolotl includes Ambystoma mexicanum, but is not limited thereto, and may also include other species of the order Caudata in the class Amphibia.

[0012] Ambystoma mexicanum (scientific name: Mexican axolotl ) is an amphibian of the family Ambystomatidae and the genus Ambystoma, also known as the axolotl, the Mexican walking fish, and the Mexican salamander. The wild Ambystoma mexicanum has a dark brown body color with black spots. Its head is relatively wide, its eyes are small, and its limbs and feet are petite. Its body length is usually 20-30 cm and its weight is 60-110 g. Its skin is smooth and scaleless, without a tympanic cavity and tympanic membrane. It has obvious external gills and a fin-like tail, which is feather-shaped. The dorsal fin extends from the back of the head to the end of the tail, and the ventral fin extends from between the two hind limbs to the end of the tail, facilitating swimming in water. Ambystoma mexicanum has become a research hotspot due to its extremely strong regenerative ability.

[0013] At present, the research on the skin of amphibious caudate species is still in its infancy, and the extraction methods of their active ingredients mostly draw on those of other amphibians (such as frogs and toads). In the existing technology, for the extraction and separation of active extracts from animal skin, the following methods and technical routes are usually adopted: (1) Solvent extraction method: This is the most traditional and widely used method. Using water, ethanol or other organic solvents as extraction media, by soaking skin tissues in the solvent, the active ingredients are released by molecular solubility. The advantage of this method is its simple operation and low cost, but it is not very friendly to heat-sensitive substances or components with easily damaged structures. (2) Enzymatic extraction method: By adding specific enzymes (such as proteases, cellulases) to selectively degrade skin tissues, so as to release proteins, polypeptides or other macromolecular active substances in the skin. Enzymatic extraction has the characteristics of strong specificity and high extraction rate, but for large-scale operations, there are problems in achieving stability, and it is easy to cause partial changes in the structure of the target protein. (3) Ultrasonic-assisted extraction: Ultrasonic waves rupture tissue cells through acoustic cavitation, enabling the active ingredients inside the cells to be quickly released into the extraction medium. This method has a short operation time and high extraction efficiency, especially suitable for the preliminary separation step. However, in practical applications, the intensity and treatment time of ultrasonic waves need to be strictly controlled, otherwise it may cause the degradation of active substances. (4) Mechanical crushing and centrifugation method: Mechanical crushing technology physically crushes skin samples through a homogenizer or tissue grinder to expose and release cell contents, and then combines high-speed centrifugation technology to separate the precipitate from the supernatant. This method is flexible in operation and suitable for small-scale extraction, but a large amount of impurities may be mixed in during the process and further purification is required. (5) Membrane separation and chromatography technology: For the further purification of crude extracts, membrane separation technology and chromatography technology (such as high-performance liquid chromatography HPLC) are widely used. Membrane separation removes macromolecular impurities through selective filtration, while chromatography technology can separate and purify target components with specific molecular structures. However, these methods are usually costly and difficult to achieve on a large scale. (6) Freeze-drying and stabilization treatment: In order to better preserve active ingredients, some existing technologies will freeze-dry the product after extraction, remove moisture at low temperature, thereby improving the stability of the extract. The use of stabilizers (such as antioxidants) can further extend the storage time of the extract, especially the activity of heat-sensitive substances.

[0014] The above-mentioned various methods provide basic support for the research of active extracts from amphibian skin. However, due to the particularity of the skin of urodele species, the existing technologies are mostly general extraction processes, and no optimized scheme specifically for the specific components of urodele skin has been formed. Moreover, there are still many deficiencies in the extraction efficiency, component stability, and protection of target active substances when using methods such as solvent extraction, enzymatic hydrolysis, and physical separation. This leads to low extraction efficiency, limited yield of active components, and problems such as easy degradation of proteins, mixing of impurities, and unsuitability for large-scale production during the extraction process, greatly restricting the practical application scope of skin active extracts from salamanders, axolotls, etc. Therefore, it is particularly important to develop a set of efficient extraction and separation schemes for the skin characteristics of urodele species, improve the separation and purification efficiency of active components, protect the stability of target active substances, optimize the extraction process and operation adaptability, and achieve large-scale and industrial production.

[0015] Based on this, in order to solve the problems such as protein degradation during the skin extraction process of amphibious urodele species such as salamanders and axolotls, protein structure changes caused by enzymatic hydrolysis extraction, excessive impurity content during the extraction process, and the nature of the extract being unsuitable for human application, the embodiments of the second aspect of the present invention provide the above-mentioned method for preparing axolotl skin extract, including the steps:

[0016] S100. Sampling the axolotl skin;

[0017] S200. Rapidly freezing the obtained skin sample with liquid nitrogen and performing freeze-thaw;

[0018] S300. Mechanically crushing the sample after freeze-thaw;

[0019] S400. Centrifugally filtering the crude extract of the sample after mechanical crushing;

[0020] S500. Ultrasonically crushing the sample extract after centrifugal filtration;

[0021] S600. Sterilizing and filtering the secondary extract after ultrasonic crushing to obtain axolotl skin extract.

[0022] The preparation method according to the second aspect embodiment of the present invention has at least the following beneficial effects: In view of the skin characteristics of amphibious caudate species, the present invention has developed an efficient, stable and sustainable optimized extraction scheme, integrating the whole process technology from sample processing to extraction and separation, ensuring the efficient separation and activity retention of the extract. The method for extracting proteins from the skin tissue of caudate species provided by the present invention includes steps such as skin sampling, sample freeze-thawing, mechanical crushing, centrifugal filtration, ultrasonic crushing, and sterilization filtration, and can extract a large amount of proteins for species such as salamanders and axolotls. First, when processing skin samples, liquid nitrogen quick-freezing treatment is adopted to achieve rapid freezing of the samples to inhibit the growth of microorganisms and the activity of enzymes, thereby effectively avoiding protein degradation. The mechanical crushing method is used to replace the enzymatic extraction method to maintain the integrity of the protein structure, and at the same time significantly increase the protein content, which is suitable for large-scale operation requirements. The ultrasonic crushing method is used to further remove impurities, and sterilization filtration is carried out to completely remove impurities, thereby improving the purity of the extract. At the same time, during the extraction process, the pH value of the extract is adjusted from weakly alkaline to weakly acidic to enhance the compatibility and adaptability to human skin. Finally, the optimized extraction and separation method proposed by the present invention, in view of the tissue characteristics of the skin of caudate species, through rapid freeze-thaw treatment of the samples, combined with mechanical crushing, ultrasonic-assisted separation and multiple filtration steps, gradually extracts the active ingredients in the skin of caudate species such as salamanders and axolotls under low-temperature conditions throughout the process, avoiding the negative impacts of enzymes, organic solvents or high temperature on the activity of the target product, and various composite proteins can be obtained. At the same time, the pH value of the buffer solution is adjusted to further optimize the properties of the extract, making it more suitable for the use requirements of human skin, and it is sterile and can be directly used as a product raw material. The above extraction technology is green, environmentally friendly and sustainable, can realize large-scale and industrial production, can significantly promote the industrial application of skin extracts of caudate species such as salamanders and axolotls, and is of great significance for promoting the development of this field. It can not only fill the gaps in the current technical field, but also provide scientific support for technological innovation in multiple fields.

[0023] In some embodiments of the present invention, the preparation method can also be applied to extract skin active ingredients of other amphibious caudate species.

[0024] Specifically, the preparation method can also be applied to the preparation of salamander skin extract, including the steps:

[0025] S10. Sampling the skin of the salamander;

[0026] S20. Performing liquid nitrogen quick-freezing and freeze-thawing on the obtained skin sample;

[0027] S30. Mechanically crushing the freeze-thawed sample;

[0028] S40. Centrifugally filtering the crude extract of the sample after mechanical crushing;

[0029] S50. Ultrasonically disrupt the sample extract after centrifugal filtration;

[0030] S60. Sterilize and filter the secondary extract after ultrasonic disruption to obtain the axolotl skin extract.

[0031] In some embodiments of the present invention, after sampling the axolotl skin, the obtained skin sample is stored in a buffer containing a protease inhibitor.

[0032] In some embodiments of the present invention, the pH value of the buffer is 5.5 - 6.

[0033] Specifically, the buffer formulation is: phosphate buffer containing 1× protease inhibitor, with a pH of 6.0. Compared with other extraction methods, the present invention adds a protease inhibitor to prevent protein degradation and obtain a high content of protein.

[0034] In some embodiments of the present invention, step S100 specifically includes:

[0035] S101. Anesthetize and euthanize the axolotl;

[0036] S102. Skin collection: Lay the euthanized axolotl flat on the table, make vertical cuts along both sides of the spinal column on the back with a scalpel, make a horizontal cut around the neck, then use forceps to peel the skin, and weigh the axolotl skin after peeling.

[0037] S103. Cleaning: Immerse the axolotl skin in phosphate buffer for cleaning, and clean it at least 3 times;

[0038] S104. Cutting into pieces: Blot the surface moisture of the cleaned axolotl skin with absorbent paper, put it into a petri dish, and cut it into small pieces of 0.3×0.3 cm size with scissors;

[0039] S105. Preservation: Transfer the small pieces of axolotl skin to a clean centrifuge tube and add buffer equal in weight to the axolotl skin.

[0040] In step S104, cutting into small pieces is to ensure sufficient quick-freezing of the axolotl skin in liquid nitrogen.

[0041] In the conventional method, the final extract is weakly alkaline, and its suitability for direct use on human skin is poor and it is difficult to be used as a cosmetic raw material. By adjusting the pH value of the phosphate buffer in the present invention, the pH value of the extract is adjusted from weakly alkaline to weakly acidic, making it more suitable for use as a cosmetic raw material and improving its compatibility with human skin. Compared with adjusting the pH value after extraction, adjusting the pH value before extraction in the present invention can prevent damage to the extract during the adjustment process.

[0042] In some embodiments of the present invention, step S200 specifically includes:

[0043] S201, liquid nitrogen quick-freezing: Immerse the centrifuge tube containing small pieces of axolotl skin into liquid nitrogen until the small pieces of axolotl skin and the buffer are completely frozen;

[0044] S202, freeze-thaw: Place the sample after liquid nitrogen quick-freezing in a -80°C refrigerator, store it for 24 hours, take it out and let it thaw at room temperature until completely thawed; then put it back into the -80°C refrigerator, store it for 24 hours, take it out and let it thaw at room temperature until completely thawed; then put it back into the -80°C refrigerator, store it for 24 hours, take it out and let it thaw at room temperature until completely thawed, and then carry out the extraction process.

[0045] During the skin extraction process, to prevent protein degradation during the sample pretreatment process, the present invention uses phosphate buffer and adds protease inhibitor at a concentration of 1×, combined with immersion liquid nitrogen quick-freezing treatment, to achieve rapid freezing of the sample to inhibit the growth of microorganisms and the activity of enzymes, thereby effectively avoiding protein degradation and ensuring the preservation of protein activity.

[0046] In some embodiments of the present invention, step S300 specifically includes:

[0047] S301, transfer the completely thawed sample to a clean centrifuge tube, and use a handheld high-speed homogenizer for mechanical crushing. The whole crushing process is carried out on ice;

[0048] S302, place the centrifuge tube on ice and let it stand still, waiting for centrifugation.

[0049] Specifically, the sample does not exceed 1 / 2 of the volume of the centrifuge tube to prevent liquid splashing during the mechanical crushing process.

[0050] Specifically, the mechanical crushing parameters: crush for 15 minutes, stop once every 5 minutes, take out the meat tendons wound around the probe head, discard them. The whole crushing process is carried out on ice, and the temperature is controlled to prevent protein denaturation.

[0051] Most conventional methods use biological enzyme dissociation, and the skin sample is prone to cause changes in protein structure during the enzymatic extraction process; moreover, this method takes a long time and is not suitable for large-scale operations. The solution adopted by the present invention is: replacing the enzymatic extraction method with the mechanical crushing method to maintain the integrity of the protein structure, and at the same time significantly increase the protein content, which is suitable for large-scale operation requirements (50 - 100 axolotls or axolotl skins can be extracted at one time); moreover, biological enzyme inactivation may require a high temperature of 60°C, which will affect the protein structure. The present invention uses mechanical crushing, operates on ice throughout the process, and controls the temperature to prevent protein denaturation.

[0052] In some embodiments of the present invention, step S400 specifically includes:

[0053] S401. Primary centrifugation: Centrifuge at 4°C and 4000g for 10 minutes;

[0054] S402. Transfer the supernatant: Use a Pasteur pipette to pierce through the upper layer of impurities and foam, take the middle layer of supernatant, and transfer it to a clean centrifuge tube;

[0055] S403. Secondary centrifugation: Centrifuge at 4°C and 4000g for 10 minutes;

[0056] S404. Filtration: Take the supernatant and filter it using a 40μm cell sieve, and collect the filtrate into a clean centrifuge tube;

[0057] S405. Re - filtration: Filter the collected filtrate again using a 10μm filter mesh, and collect the liquid into a clean centrifuge tube for ultrasonic disruption preparation.

[0058] Centrifuging the crude extract is to remove larger impurities. By centrifuging twice, impurities can be removed simply and quickly without the need for salting - out precipitation or ultrafiltration; while the two - stage filtration can fully filter out obvious impurities visible to the naked eye, waiting for further removal of minute impurities by ultrasonic disruption.

[0059] In some embodiments of the present invention, step S500 specifically includes:

[0060] S501. Transfer the liquid to a beaker and place the beaker on ice;

[0061] S502. Ultrasonic disruption: Set the disruption power to 1%, the ultrasonic on - time to 4.0s, the ultrasonic off - time to 6.0s, the working time to 30 minutes, control the temperature below 35°C, immerse the probe 0.5 cm from the bottom of the beaker, and perform the ultrasonic process on ice;

[0062] S503. After the ultrasonic disruption is completed, transfer the liquid to a clean centrifuge tube.

[0063] During ultrasonic disruption, the liquid in the beaker does not exceed 1 / 2 of the beaker volume (for example, for a 50mL beaker, the liquid volume is controlled at 20 - 25mL).

[0064] The crude extract contains a relatively large amount of impurities, which affects subsequent applications. The present invention uses ultrasonic disruption to further remove impurities, and the whole process is operated on ice to ensure that the protein will not denature due to high temperature. After disruption, filter twice using a 0.22μm filter mesh to completely remove impurities, thereby improving the purity of the extract.

[0065] In some embodiments of the present invention, the secondary extract after ultrasonic disruption is subjected to double sterile filtration, and the filter membrane aperture used for the double sterile filtration is 0.22μm.

[0066] In some embodiments of the present invention, step S600 specifically includes:

[0067] S601. Centrifuge the liquid after ultrasonic disruption at 4°C and 4000 g for 10 minutes, and transfer the supernatant to a clean centrifuge tube.

[0068] S602. Vacuum filtration: Filter using a vacuum filter / 0.22 µm pore size / PES filter membrane. Connect the filter flask to the vacuum pump, pour the supernatant into the filter flask, and start the vacuum pump for filtration.

[0069] S603. Filtration: Filter again using a 0.22 µm pore size / CA filter membrane syringe filter. Connect the syringe to the needle filter, pour the liquid into the syringe, push for filtration, and collect the filtered liquid into a clean centrifuge tube.

[0070] S604. Preservation: Store the collected axolotl skin extract at -80°C.

[0071] The present invention adopts double sterilization filtration with a filter membrane pore size of 0.22 µm, and the obtained axolotl extract is free of impurities and bacteria, ensuring the usability of the extract.

[0072] In some embodiments of the present invention, after step S600, step S700 is further included: BCA protein determination.

[0073] Specifically, step S700 includes:

[0074] S701. Dilution: Take the axolotl skin extract and perform 10-fold, 20-fold, and 30-fold dilutions.

[0075] S702. Use a BCA protein concentration determination kit to determine the protein concentration.

[0076] S703. Calculate the protein concentrations of the 10-fold, 20-fold, and 30-fold dilutions and take the average value.

[0077] In summary, for the method of extracting axolotl skin provided by the present invention, during the sample processing, to prevent protein degradation, phosphate buffer solution is used and protease inhibitor is added at a concentration of 1×. The effect of rapid freezing is achieved through immersion in liquid nitrogen, thereby inhibiting the growth of microorganisms and the activity of enzymes, and ensuring the preservation of protein activity. Aiming at the problems that the traditional enzymatic hydrolysis extraction method is prone to cause changes in protein structure and is not suitable for large-scale operation, the mechanical crushing method is adopted. By physical crushing means, the protein content is kept high and the structure is complete, while the process flow is simplified and the industrial operability is improved. To further remove impurities in the extract, the present invention combines ultrasonic treatment technology. By operating on ice throughout the process, it is ensured that the protein does not denature due to high temperature. The extract after ultrasonic treatment is filtered through a 0.22μm filter twice to remove fine impurities and improve the purity of the extract. At the same time, to improve the chemical properties of the extract to adapt to human skin application, the present invention adjusts the pH value of the phosphate buffer solution from weakly alkaline to weakly acidic, making the final product more suitable as a cosmetic raw material to meet the needs of human skin care. At the same time, the above skin extraction method is also applicable to other species of the order Caudata such as salamanders.

[0078] Through the above innovative process, the present invention realizes a stable preparation method for skin extracts applicable to species of the order Caudata. The whole process adopts physical extraction means, avoiding the damage to the quality and activity of the extract caused by enzymatic hydrolysis or chemical reagents. At the same time, fully considering the structural characteristics of the skin of Caudata such as salamanders and axolotls, impurities are removed and the product properties are optimized through precise multi-step processing, and a highly pure, highly active and sterile skin extract is successfully obtained. The process of the present invention has passed a number of functional verification experiments, proving its high stability and applicability, and showing broad application potential.

[0079] An embodiment of the third aspect of the present invention provides an application of the above axolotl skin extract or the axolotl skin extract obtained by the above preparation method in skin anti-aging, and the application is for non-diagnostic or therapeutic purposes.

[0080] According to the application of the third aspect of the present invention, there are at least the following beneficial effects: the skin extract of the amphibian provided by the present invention contains rich bioactive ingredients, such as collagen (COL) family members, fibrin (FCN) family members, C1Q complement protein family members, fibronectin (FN1), cytochrome b5 reductase 3 (CYB5R3), nicotinamide phosphoribosyltransferase (NAMPT), or mitochondrial acetaldehyde dehydrogenase 2 (ALDH2), and the above ingredients interact and promote each other, giving the extract effective skin anti-aging function. And unlike most anti-aging products with a single effect, it has multiple dimensions of skin anti-aging effects, such as significant anti-wrinkle, firming, moisturizing, soothing, oil control, anti-photoaging and other effects. The above multi-dimensional anti-aging scheme can work together for fundamental regulation, with strong effectiveness and good long-lasting effect. Secondly, the chemical properties of the extract of the present invention are adapted to human skin, its pH is naturally compatible with the skin microecology, can reduce side effects or adverse reactions, has good safety, high affinity, and can meet the application needs of human skin. In addition, the above application scheme also provides another idea for developing more safe, effective and long-lasting anti-aging products. It has great application potential. It can not only be used to develop skin care products and drugs based on the extract to delay skin aging or treat aging-related skin diseases, but also can be used to develop skin repair drugs. It is even expected to realize personalized anti-aging plans, which has extremely high scientific research significance and practical value.

[0081] In some embodiments of the present invention, the anti-aging includes at least one of anti-wrinkle, firming, moisturizing, soothing, oil control, or anti-photoaging.

[0082] An embodiment of the fourth aspect of the present invention provides an application of the above-mentioned Ambystoma skin extract or the Ambystoma skin extract obtained by the above-mentioned preparation method in skin damage repair, wherein the application is for non-diagnostic or therapeutic purposes.

[0083] The fifth aspect of the present invention provides an embodiment of the use of the above-mentioned Ambystoma skin extract or the Ambystoma skin extract obtained by the above-mentioned preparation method in at least one of (a1) to (a7), wherein the use is for non-diagnostic or therapeutic purposes:

[0084] (a1) developing and / or preparing skin anti-aging products;

[0085] (a2) developing and / or preparing skin care products;

[0086] (a3) developing and / or preparing drugs for treating aging-related skin diseases;

[0087] (a4) Developing and / or preparing drugs for repairing skin damage;

[0088] (a5) constructing a skin aging model;

[0089] (a6)Construct a skin injury repair model;

[0090] (a7)Develop and / or prepare elastase inhibitors, hyaluronidase inhibitors, 5α-reductase inhibitors, or free radical scavengers.

[0091] In some embodiments of the present invention, the applications include developing and / or preparing skin anti-aging products or skin care products. The axolotl skin extract of the present invention, due to its sterile, high-purity, and weakly acidic properties, can effectively meet the needs of human skin and is an ideal raw material for cosmetics or skin care products. Its antioxidant and moisturizing functions can be used in skin care products such as facial masks, serums, and lotions to provide anti-aging, sunscreen repair, and deep moisturizing effects. In addition, its antibacterial properties are also suitable for the development of functional cosmetics for acne treatment, improving sensitive skin, and repairing the skin barrier.

[0092] In some embodiments of the present invention, the applications include developing and / or preparing drugs for treating skin diseases related to aging or skin injury repair drugs. The axolotl skin extract of the present invention contains rich bioactive components, including polypeptides and proteins with antibacterial, anti-inflammatory, antioxidant, and other functions. In the medical field, these components can be used to develop new antibacterial drugs, tissue repair agents, and skin healing products. For example, in wound repair, the extract provides an effective approach for the development of drugs for burns, postoperative wounds, and chronic skin ulcers by promoting collagen production, inhibiting inflammatory responses, and microbial infections. At the same time, the extract has potential value in the development of anti-aging drugs, capable of slowing down the process of cell aging and enhancing cell activity.

[0093] In some embodiments of the present invention, the applications include developing and / or preparing functional foods or health products.

[0094] Due to the natural source and biological activity of the axolotl skin extract, the products of the present invention have the potential to be used as raw materials for functional foods and health products. Its antioxidant properties help reduce the damage of free radicals to the body, thereby enhancing immunity and delaying aging. In health foods, the extract can be made into capsules, beverages, or oral liquids to help improve skin health and enhance the body's defense mechanism.

[0095] In some embodiments of the present invention, the applications include developing biomaterials.

[0096] The high activity and purity of the amphisbaenian skin extract of the present invention make it promising in the field of biomaterial development. For example, it can be used as a new type of bio-glue or coating material, applied to the surface of medical implants and medical devices to improve antibacterial properties and biocompatibility. In addition, some active ingredients in the extract may have good tissue engineering properties and can be used for 3D printing scaffold materials to promote tissue regeneration and repair.

[0097] In some embodiments of the present invention, the application includes constructing a drug screening platform.

[0098] The high-purity Ambystoma skin extract provided by the present invention can also provide high-quality materials for biomedical and biochemical research, and its active ingredients can be used to study cell signaling pathways, protein structure-function relationships, and the mechanism of action of bioactive molecules. At the same time, the extract can be used in a high-throughput screening platform for new drugs to help quickly discover and verify new drug targets with clinical value.

[0099] The above applications provide a solid foundation for technological progress in multiple fields and demonstrate broad market value and social benefits.

[0100] An embodiment of the sixth aspect of the present invention provides a product, comprising the above-mentioned Ambystoma salamander skin extract or the Ambystoma salamander skin extract obtained by the above-mentioned preparation method.

[0101] In some embodiments of the present invention, the product comprises a medicine, a pharmaceutical composition, a skin care product, or a cosmetic.

[0102] The product provided by the present invention includes the above-mentioned Ambystoma skin extract as an active ingredient, and thus has all the technical effects of the above-mentioned extract. The anti-aging or skin damage repair-related drugs, pharmaceutical compositions, skin care products, or cosmetics developed based on the extract can provide skin anti-aging effects in multiple dimensions (for example, significant anti-wrinkle, firming, moisturizing, soothing, oil control, anti-photoaging and other effects) through the mutual complementation and mutual promotion between the rich biologically active ingredients contained therein, and effectively repair skin damage (including skin damage caused by diseases, trauma, etc.). The above-mentioned multi-dimensional anti-aging scheme and damage repair scheme can be fundamentally adjusted, with strong effectiveness and good long-lasting effect. Moreover, the chemical properties of the extract of the present invention are adapted to human skin, its pH is naturally compatible with the skin microecology, can reduce side effects or adverse reactions, has good safety, high affinity, and can meet the application needs of human skin. In addition, the above application scheme also provides another idea for developing more safe, effective and long-lasting products, and has great application potential. It can not only be used to develop skin care products and drugs based on the extract to delay skin aging or treat aging-related skin diseases, but also can be used to develop skin repair drugs, and even has the potential to realize personalized anti-aging plans, which has extremely high scientific research significance and practical value.

[0103] In some embodiments of the present invention, the drug, pharmaceutical composition, skin care product, or cosmetic further comprises a pharmaceutically or cosmetically acceptable carrier.

[0104] In some embodiments of the present invention, the skin care product or cosmetic includes anti-wrinkle products, firming products, moisturizing products, soothing products, oil-control products, and anti-photoaging products.

[0105] In some embodiments of the present invention, the categories of the skin care product or cosmetic include essence, lotion, cream, and mask.

[0106] In some embodiments of the present invention, the ingredients of the skin care product or cosmetic further include at least one of glycerol, hyaluronic acid, propylene glycol, or butylene glycol.

[0107] In some embodiments of the present invention, the product includes at least one of the following (b1) to (b8):

[0108] (b1) Skin anti-aging product;

[0109] (b2) Skin care product;

[0110] (b3) Drug for treating skin diseases related to aging;

[0111] (b4) Drug for repairing skin damage;

[0112] (b5) Elastase inhibitor;

[0113] (b6) Hyaluronidase inhibitor;

[0114] (b7) 5α-reductase inhibitor;

[0115] (b8) Free radical scavenger;

[0116] The active ingredient of the product includes the axolotl skin extract described above or the axolotl skin extract obtained by the preparation method described above.

[0117] In some embodiments of the present invention, the product is a topical preparation prepared with the axolotl skin extract as the active ingredient and adding pharmaceutically or cosmetically acceptable excipients or auxiliary ingredients.

[0118] In some embodiments of the present invention, the topical preparation includes cosmetics or skin care products.

[0119] In some embodiments of the present invention, the cosmetics or skin care products include anti-wrinkle cream, firming cream, moisturizing lotion or milk, soothing gel or jelly, oil-control cream or milk, and anti-photoaging cream or powder.

[0120] In some embodiments of the present invention, the types of the cosmetics or skin care products include aqueous solutions, emulsions, creams, powders, gels, jellies, essences, or facial masks.

[0121] In some embodiments of the present invention, the acceptable excipients include at least one of diluents, binders, wetting agents, humectants, thickeners, solvents, emulsifiers, cosolvents, preservatives, pH regulators, osmotic pressure regulators, surfactants, and buffers.

[0122] In some embodiments of the present invention, the auxiliary components include at least one of whitening agents, emollients, anti-acne agents, ultraviolet absorbers, and skin conditioners.

[0123] In some embodiments of the present invention, the auxiliary components further include at least one of collagen, vitamins, tea polyphenols, and coenzyme Q10.

[0124] Other features and advantages of the present invention will be set forth in the following description, and in part will be obvious from the description, or may be learned by practice of the present invention. The objectives and other advantages of the present invention may be realized and attained by the structure particularly pointed out in the specification, claims, as well as the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 Schematic diagram of the BCA concentration measurement results provided by the embodiments of the present invention;

[0126] Figure 2 Schematic diagram of the detection results of the anti-wrinkle and firming effects of the axolotl skin extract provided by the embodiments of the present invention;

[0127] Figure 3 Schematic diagram of the detection results of the moisturizing effect of the axolotl skin extract provided by the embodiments of the present invention;

[0128] Figure 4 Schematic diagram of the detection results of the soothing effect of the axolotl skin extract provided by the embodiments of the present invention;

[0129] Figure 5 Schematic diagram of the detection results of the oil control effect of the axolotl skin extract provided by the embodiments of the present invention;

[0130] Figure 6 Schematic diagram of the detection results of the anti-photoaging effect of the axolotl skin extract provided by the embodiments of the present invention;

[0131] Figure 7 Schematic diagram of the evaluation results of the skin damage repair effect of the axolotl skin extract provided by the embodiments of the present invention: the wound morphology of the control group on the 20th day;

[0132] Figure 8Schematic diagram of the evaluation result of the skin damage repair effect of the axolotl skin extract provided by the embodiment of the present invention: the wound morphology of the experimental group on the 20th day. Detailed implementation manners

[0133] The following will clearly and completely describe the concept and technical effects of the present invention in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0134] In the description of the present invention, the description of reference terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0135] In the description of the present invention, unless otherwise specified, the numerical range "a~b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. Unless otherwise specified, each reaction or operation step can be carried out in sequence or not in sequence. Preferably, the reaction method in the present invention is carried out in sequence.

[0136] The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in this embodiment are, unless otherwise specified, reagents and materials obtained from commercial channels.

[0137] Example 1 Preparation of axolotl skin extract

[0138] 1) Purchase 35 white Mexican axolotls with a size of 15 - 17 cm from a farm with artificial breeding qualifications (Jiutai District Kalun Ornamental Fish Farm, Group 6, Kalun Sunjia Village, Jiutai District, Changchun City, Jilin Province).

[0139] 2) Before sampling, anesthetize the axolotl using the fish anesthetic ethyl 3-aminobenzoate methanesulfonate (J&K Scientific, 247097). Anesthesia protocol: Add 1.5 g of the anesthetic to 1 L of water, place the axolotl in it, and anesthetize for 15 minutes until the body is immobile. Then sacrifice and take the skin: Lay the axolotl flat on the table, make vertical incisions along both sides of the spinal column on the back with a scalpel, and make a horizontal incision around the neck. Use forceps to peel off the skin. Finally, obtain approximately 170 g of axolotl skin.

[0140] 3) Immerse the axolotl skin in phosphate buffer and wash it 3 times.

[0141] 4) After washing, use absorbent paper to blot the water on the surface of the axolotl skin.

[0142] 5) Place it in a 10 cm petri dish, cut it into small pieces of 0.3×0.3 cm size with scissors (ensure that the axolotl skin is fully frozen in liquid nitrogen), transfer it to a 15 ml centrifuge tube, and add phosphate buffer (pH = 6.09) containing 1× protease inhibitor equal to the weight of the skin.

[0143] 6) Immerse the 15 mL centrifuge tube containing axolotl skin pieces in liquid nitrogen for 5 minutes until the axolotl skin pieces and the buffer are completely frozen.

[0144] 7) After quick freezing, place it in a -80°C refrigerator. After freezing for 24 hours, take it out and thaw at room temperature.

[0145] 8) Put it back into the -80°C refrigerator again, freeze for 24 hours, and take it out and thaw at room temperature.

[0146] 9) Repeat the operation in step 8 once.

[0147] 10) After complete thawing, transfer the sample to a clean 50 mL centrifuge tube, and the liquid level should not exceed 25 mL to prevent liquid splashing during mechanical fragmentation. Use a handheld high-speed homogenizer to crush it. Mechanical fragmentation parameters: Crush for 15 minutes, stop once every 5 minutes, take out the meat tendons wrapped around the probe head, discard them, and perform the entire crushing process on ice to control the temperature so that the protein does not denature.

[0148] 11) After complete crushing, centrifuge at 4°C, 4000 g for 10 minutes.

[0149] 12) Use a Pasteur pipette to pierce through the upper layer of impurities and foam, take the middle layer of supernatant, transfer it to a clean 50 mL centrifuge tube, and centrifuge again at 4°C, 4000 g for 10 minutes.

[0150] 13) Transfer the supernatant for filtration, filter it using a 40 μm cell sieve, and collect the filtrate into a clean 50 mL centrifuge tube.

[0151] 14) Filter again using a 10-μm filter and collect the liquid in a clean 50-mL centrifuge tube.

[0152] 15) Transfer the filtered axolotl skin extract to a 50-mL beaker, controlling the liquid volume at 20 - 25 mL, and place the beaker on ice.

[0153] 16) Set the crushing power to 1%, the ultrasonic on-time to 4.0 s, the ultrasonic off-time to 6.0 s, the working time to 30 minutes, control the temperature below 35°C, immerse the probe 0.5 cm from the bottom of the beaker, and perform the ultrasonic process on ice.

[0154] 17) After ultrasonic crushing is completed, transfer the liquid to a clean 50-ml centrifuge tube, centrifuge at 4°C and 4000 g for 10 minutes. At this time, there is an obvious black precipitate at the bottom. After centrifugation, transfer the supernatant to a new 50-mL centrifuge tube.

[0155] 18) Filter using a Corning 500 mL vacuum filter / 0.22 µm pore size / PES filter membrane. Connect the filter flask to the vacuum pump, pour the liquid into the filter flask, start the vacuum pump, and perform filtration to collect the filtered liquid.

[0156] 19) Filter again using a 0.22 µm pore size / CA filter membrane syringe filter. Connect the syringe to the needle filter, pour the liquid into the syringe, push for filtration, and collect the filtered liquid into a clean 50-mL centrifuge tube to obtain a total of 140 ml of extract liquid.

[0157] 20) Measure the BCA concentration using a BCA Protein Assay Kit (Enhanced).

[0158] Some of the reagents and manufacturers used in this example are shown in Table 1:

[0159] Table 1

[0160]

[0161] The BCA concentration measurement results are as Figure 1 and Table 2. It can be seen that the protein concentration of the extract is 18.4 mg / mL, the total volume of the extract is 140 mL, and the total amount of protein is 2576 μg.

[0162] Table 2

[0163]

[0164] Example 2 Sterility Test of Axolotl Skin Extract

[0165] Take 1 mL of the axolotl skin extract of Example 1 for sterility testing. The testing items are as follows: Klebsiella pneumoniae, Salmonella, Escherichia coli, Bordetella bronchiseptica, Pasteurella multocida, Pasteurella pneumotropica, Group B Streptococcus, Streptobacillus moniliformis, Corynebacterium muris, Staphylococcus aureus, Pseudomonas aeruginosa. The test results are all sterile.

[0166] Activity substance detection of the axolotl skin extract of Example 3

[0167] Take 10 mL of the axolotl skin extract of Example 1 for mass spectrometry detection, and confirm the contained proteins according to the peptide map. The specific method is as follows: The corresponding peptide segments are cleaved at the peptide bond position by ETD (electron transfer dissociation), and the corresponding b-ions and y-ions will be generated. Each b / y ion has a corresponding mass-to-charge ratio (mass divided by charge) and a corresponding signal intensity, generating a theoretical mass spectrum (the x-axis is the mass-to-charge ratio and the y-axis is the signal intensity). By matching with the theoretical spectrum, the detected b / y ions are determined, and thus the peptide sequence corresponding to the actual spectrum is inferred.

[0168] After analysis, the axolotl skin extract contains a variety of active ingredients, including: various in the collagen family, such as COL1A1 protein, COL1A2 protein, COL3A1 protein, COL4A1 protein, COL5A1 protein, COL5A2 protein, COL6A1 protein, COL6A2 protein, COL6A3 protein, COL6A6 protein, COL11A1 protein, COL12A1 protein, COL17A1 protein, COL18A1 protein, COL28A1 protein, COLGALT1 protein, etc.; various in the fibrinogen family, such as FCN1 protein, FCN3 protein, FCN2 protein, etc.; various in the C1Q complement protein family, such as C1QC protein, C1QB protein, C1QA protein, C1QTNF3 protein, etc.; fibronectin (FN1); cytochrome b5 reductase 3 (CYB5R3); nicotinamide phosphoribosyltransferase (NAMPT); mitochondrial aldehyde dehydrogenase 2 (ALDH2). (The protein mass spectrometry detection results are not shown)

[0169] Efficacy detection and safety evaluation of the axolotl skin extract of Example 4

[0170] Take 40 mL of the original solution of the axolotl skin extract in Example 1 for efficacy and safety evaluation. The detection items include anti-wrinkle, firming, moisturizing, soothing, oil control, and anti-photoaging efficacy evaluations. The detection basis includes the laboratory method LC-WI-HZ-176 Operating Instruction for Elastase Inhibition Test, LC-WI-HZ-180 Experimental Instruction for Moisturizing Efficacy Test, LC-WI-HZ-185 Operating Instruction for Hyaluronidase Inhibition Experiment, LC-WI-HZ-178 Operating Instruction for 5α-Reductase Inhibition Experiment, and LC-WI-HZ-174 Operating Instruction for Free Radical (DPPH) Scavenging Experiment.

[0171] 1. Detection Results of Anti-Wrinkle and Firming Efficacy

[0172] Elastic fibers are composed of elastin and microfibrils and are distributed in the dermis and subcutaneous tissues, making the skin elastic. However, due to the action of some environmental factors such as ultraviolet irradiation, pressure, and pollution, it will promote the production of elastase in the body. Elastase is a member of the chymotrypsin family and will degrade elastin, resulting in the loss of connective tissue in the epidermis, thus leading to skin relaxation and the formation of wrinkles. Therefore, inhibiting the production of elastase can block or prevent skin relaxation and wrinkles, achieving anti-wrinkle and firming effects. In this example, the results of the elastase inhibition rate of the axolotl skin extract sample were compared with those of the blank control group. If the inhibition rate of the axolotl skin extract sample is better than (higher than) that of the blank control group and the statistical difference P value < 0.05, it can be considered that the sample has certain anti-wrinkle and firming effects.

[0173] The steps are as follows: Pretreat each group of samples to make a mixed solution; incubate in an incubator at 25 °C for 15 minutes; then add a certain amount of elastase and continue to incubate for 15 minutes; use an enzyme-labeled analyzer to measure the OD value of the elastase inhibition rate at a wavelength of 410 nm; calculate the inhibition rate and statistically analyze the difference P value.

[0174] The results are as Figure 2 shown in Table 3.

[0175] Table 3

[0176]

[0177] As can be seen from the results, the inhibition rate of the axolotl skin extract sample is better than (higher than) that of the blank control group and the statistical difference P value < 0.05, indicating that the axolotl skin extract sample has firming and anti-wrinkle effects.

[0178] 2. Detection Results of Moisturizing Efficacy

[0179] The moisture in the stratum corneum of the skin plays an important role in maintaining skin health, preventing skin aging, and moisturizing. The cause of skin dryness is insufficient moisture in the stratum corneum, and the moisture often binds to the stratum corneum in the form of bound water. In this embodiment, by continuously monitoring the change in the moisture weight of the test sample of the skin extract of the axolotl after treatment, a moisture change rate Δm - time change curve of the test sample is drawn. According to the trend of the moisture change rate Δm curve, the moisturizing ability of the test sample is observed, and then the skin moisturizing effect of the test sample is evaluated.

[0180] The steps are as follows: Pretreat the sample, place the sample and the positive control (glycerol is used in this embodiment) in a thermostatic and humidified chamber under certain temperature and humidity conditions, regularly weigh with an electronic balance, record the data, calculate the moisture change rate Δm, and make a curve.

[0181] The results are as Figure 3 shown in Table 4.

[0182] Table 4 Test results of moisture change rate Δm

[0183]

[0184] It can be seen from the results that the moisture change rate Δm curve of the axolotl skin extract sample is smaller (better) than the moisture change rate Δm curve of the positive control, indicating that the sample has a moisturizing effect.

[0185] 3. Detection results of soothing effect

[0186] Hyaluronic acid is widely present in the skin and other tissues, and the relationship between hyaluronic acid and the skin is very close. Macromolecular hyaluronic acid can inhibit the inflammatory response, thus playing a role in relieving the discomfort caused by skin inflammation. Macromolecular hyaluronic acid will be degraded under the action of hyaluronidase, resulting in a decrease in the content of macromolecular hyaluronic acid. The degradation product N-acetylglucosamine is produced by decomposition. Measuring the content of N-acetylglucosamine in the reaction system can indirectly reflect the activity of hyaluronidase. In this embodiment, the inhibition rate of the test sample on hyaluronidase is determined by measuring the content of N-acetylglucosamine in the reaction system. If the inhibition rate of the test sample is better (higher) than the inhibition rate of the blank control group and the statistical difference P value < 0.05, it can be considered that the test sample has a certain soothing effect.

[0187] The steps are as follows: Pretreat the test sample, prepare sample tubes with different dilution degrees, and at the same time make a parallel blank group and a positive control; add reactants to each group and mix well, then place them in a constant temperature water bath at 37°C for 40 minutes; add a PH regulator, place them in a boiling water bath for 15 minutes, cool in an ice bath for 10 minutes, and then cool to room temperature; measure the OD value with an enzyme-labeled analyzer at a wavelength of 530nm, calculate the inhibition rate of hyaluronidase activity and the statistical difference P value.

[0188] The results are as Figure 4 shown in Table 5.

[0189] Table 5

[0190]

[0191] It can be seen from the results that the inhibition rate of the axolotl skin extract sample is better than (higher than) that of the blank control group, and the statistical difference P value < 0.05, indicating that the axolotl skin extract sample has a soothing effect.

[0192] 4. Detection results of oil control efficacy

[0193] 5α-reductase is a membrane protease that depends on reduced coenzyme II (NADPH) and is an important androgen metabolic enzyme in the skin. It can irreversibly convert testosterone (T) into dihydrotestosterone (DHT). DHT is the most active androgen and can induce excessive sebum secretion by sebaceous glands. By inhibiting the activity of 5α-reductase to reduce the level of DHT, the excessive sebum secretion by sebaceous glands can be effectively alleviated. In this example, the inhibition rate test results of the test sample and the blank control group were compared for 5α-reductase. If the inhibition rate of the test sample is better than (higher than) that of the blank control group and the statistical difference P value < 0.05, it can be considered that the test sample has a certain oil control efficacy.

[0194] The steps are as follows: Pretreat each group of samples, add them to the well plate for pre-reaction, and place them in an incubator at 37°C for 30 minutes; after the pre-reaction is completed, add the enzyme reagent and continue the reaction, and place it in an incubator at 37°C for 30 minutes; first add the chromogenic agent to each well, develop color in the incubator at 37°C in the dark, add the termination solution to terminate the reaction; measure the absorbance of each well by machine testing, record the data, and calculate the inhibition rate.

[0195] The results are as Figure 5 shown in Table 6.

[0196] Table 6

[0197]

[0198] It can be seen from the results that the inhibition rate of the axolotl skin extract sample is better than (higher than) that of the blank control group, and the statistical difference P value < 0.05, indicating that the axolotl skin extract sample has an oil control efficacy.

[0199] 5. Detection results of anti-photoaging efficacy

[0200] Ultraviolet radiation in sunlight can cause photoaging of human skin tissue, one of the characteristics of which is the appearance of aging and wrinkles on the skin of light-exposed areas. There are various mechanisms that cause skin photoaging. One of them is that light radiation can lead to the generation of free radicals. Free radicals have extremely strong oxidation ability. They attack any biomolecule through oxidation reactions, and undergo various oxidation reactions with macromolecular substances such as lipids, carbohydrates, proteins, and deoxyribonucleic acid, causing oxidative damage such as denaturation, cross-linking, and breakage, and then leading to the destruction of cell structure and function, as well as the damage of body tissues and the lesions of organs, which is manifested as photoaging on the skin. When there is a free radical scavenger in the test sample, the light absorption of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) reagent (a stable long-lived free radical) weakens. Thus, the ability of the test sample to scavenge free radicals, that is, the size of the antioxidant activity, can be evaluated. In this example, the DPPH free radical scavenging rate test is carried out on the test sample and the blank control group. If the scavenging rate of the test sample is better than (higher than) the scavenging rate of the blank control group and the statistical difference P value < 0.05, it can be considered that the test sample has a certain anti-photoaging effect.

[0201] The steps are as follows: Pretreat the test sample, prepare sample tubes with different dilutions, and at the same time make parallel blank groups and positive controls; add the free radical DPPH to prepare multiple tubes of samples, shake well, and set aside; after standing at room temperature for 5 minutes, use an enzyme-linked immunosorbent assay analyzer to measure the absorbance value at a wavelength of 517 nm; record the data, calculate the scavenging rate before and after adding the test substance and the statistical difference P value.

[0202] The results are as Figure 6 shown in Table 7.

[0203] Table 7

[0204]

[0205] It can be seen from the results that the scavenging rate of the axolotl skin extract sample is better than (higher than) the scavenging rate of the blank control group and the statistical difference P value < 0.05, indicating that the axolotl skin extract sample has an anti-photoaging effect.

[0206] Example 5 Evaluation of the skin damage repair effect of axolotl skin extract

[0207] In this example, the skin damage repair effect of axolotl skin extract is verified through a scald model of SD rats and an intervention experiment.

[0208] 1. Use 8-week-old SD rats and randomly and evenly divide them into groups (control group and experimental group).

[0209] 2. Preparation stage

[0210] The abdominal hair of SD rats was shaved to ensure smooth and hairless skin; a hair removal cream was used to further remove hair from the shaved area for 9 minutes; after hair removal, the shaved area was cleaned with physiological saline and dried for later use.

[0211] 3. Scald treatment

[0212] A metal hot plate with a diameter of 1.5 cm was used, and the temperature of the hot plate was adjusted to 200 °C. Each side of the lower abdomen of the SD rats was scalded once, and each scald lasted for 12 seconds; after scald treatment, the scalded area was observed in a timely manner and the wound surface was cleaned.

[0213] 4. Grouping treatment

[0214] Control group: The mask cloth was soaked in 300 μL of PBS (phosphate buffer solution), applied to the scalded wound surface, and then sterilely bandaged.

[0215] Experimental group: 300 μL of the axolotl extract obtained in Example 1 was evenly applied to the scalded wound surface, and then covered with a mask cloth and sterilely bandaged in the same way.

[0216] 5. Dressing change and observation

[0217] Week 1: The drugs and dressings were changed daily, and the wound healing situation was observed and recorded.

[0218] From week 2 to day 20: The drugs and dressings were changed every 3 days, and the wound healing progress was continuously recorded, including indicators such as wound surface shrinkage rate and healing area.

[0219] 6. Result analysis

[0220] After the experiment ended on day 20, morphological and histological analyses were performed on the wound surfaces of each group, and ImageJ software was used to compare the differences in wound healing effects between the control group and the experimental group.

[0221] On day 20, the wound surface morphologies of the control group ( Figure 7 ) and the experimental group ( Figure 8 ) were as shown in Figures 7 - 8 . The traumatic area statistics are shown in Table 8.

[0222] Table 8

[0223]

[0224] It can be seen from the results that the axolotl skin extract sample has a better efficacy in repairing skin damage than the control group, indicating that it has a good skin damage repair function. It can be used to develop skin care products and drugs based on this extract to treat or improve skin damage, and can also be used to develop skin repair drugs, which has extremely high scientific research significance and practical value.

[0225] The above is a specific description of the preferred embodiment of the present application. However, the present application is not limited to the above-mentioned implementation manners. Those skilled in the art can also make various equivalent deformations or substitutions without departing from the spirit of the present application, and these equivalent deformations or substitutions are all included within the scope defined by the claims of the present application.

Claims

1. Use of an axolotl skin extract in the preparation of a drug or cosmetic for repairing skin damage, the preparation method of the axolotl skin extract comprising the steps of: Sampling the axolotl skin, and storing the obtained skin sample in a buffer solution containing a protease inhibitor, the pH value of the buffer solution being 5.5-6; Quick-freezing the obtained skin sample with liquid nitrogen and subjecting it to freeze-thaw cycles; Mechanically crushing the sample after freeze-thaw cycles; Centrifugally filtering the crude extract of the sample after mechanical crushing; Subjecting the sample extract after centrifugal filtration to ultrasonic crushing; Sterilizing and filtering the secondary extract after ultrasonic crushing to obtain the axolotl skin extract; Wherein, the axolotl is purchased from a farm with artificial breeding qualifications.

2. The application according to claim 1, wherein The axolotl includes Ambystoma mexicanum.

3. The application according to claim 1, wherein Subjecting the secondary extract after ultrasonic crushing to double sterilizing filtration, the pore size of the filter membrane used for the double sterilizing filtration being 0.22 μm.

4. Use of an axolotl skin extract in the preparation of a product for skin anti-aging, the anti-aging including at least one of anti-wrinkle, firming, moisturizing, soothing, oil control, or anti-photoaging; The preparation method of the axolotl skin extract comprising the steps of: Sampling the axolotl skin, and storing the obtained skin sample in a buffer solution containing a protease inhibitor, the pH value of the buffer solution being 5.5-6; Quick-freezing the obtained skin sample with liquid nitrogen and subjecting it to freeze-thaw cycles; Mechanically crushing the sample after freeze-thaw cycles; Centrifugally filtering the crude extract of the sample after mechanical crushing; Subjecting the sample extract after centrifugal filtration to ultrasonic crushing; Sterilizing and filtering the secondary extract after ultrasonic crushing to obtain the axolotl skin extract; Wherein, the axolotl is purchased from a farm with artificial breeding qualifications.

5. The application according to claim 4, wherein The application includes the preparation of skin care products.

6. The application according to claim 4, characterized in that The application includes the preparation of drugs for treating skin diseases related to aging.

7. The application according to claim 4, characterized in that, The application includes the preparation of elastase inhibitors, hyaluronidase inhibitors, 5α-reductase inhibitors, or free radical scavengers.

8. The application according to claim 4, wherein The product includes a pharmaceutical composition or a cosmetic.

9. The application according to claim 8, characterized in that, The pharmaceutical composition or the cosmetic further includes a pharmaceutically or cosmetically acceptable carrier.

10. The application according to claim 8, wherein The cosmetic includes anti-wrinkle products, firming products, moisturizing products, soothing products, oil control products, anti-photoaging products; And / or, the categories of the cosmetic include essence, lotion, cream, mask; And / or, the ingredients of the cosmetic further include at least one of glycerol, hyaluronic acid, propylene glycol, or butanediol.

Images