Spirit skin extract as well as preparation method and application thereof

Abstract

The invention discloses a spirulina platense skin extract as well as a preparation method and application thereof, and relates to the technical field of biomedicine. The obscured spirulina skin extract is rich in various active ingredients including collagen family members, viscose protein, C1Q complement protein, fibronectin and the like, and the ingredients complement and promote one another, so that the extract has remarkable effects in the aspects of skin aging resistance, skin injury repair and the like; therefore, the method has a wide application prospect in medicine research and development, cosmetics and functional foods. According to the extraction method of the skin protein of the obtuse spirulina, the sample is subjected to rapid freeze thawing treatment, and the steps of mechanical crushing, ultrasonic-assisted separation and multiple filtration are combined, so that the influence on the activity of the product is avoided, and various compound proteins can be obtained; the pH value of a buffer solution is adjusted, the property of the extract is further optimized, and the extract is more suitable for human skin, is sterile and can be directly used as a product raw material, so that large-scale production is realized, and industrial application of the obtuse spirulina skin extract is promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an amphibian skin extract and a preparation method and application thereof. Background Art

[0002] With the continuous advancement of biotechnology and natural resource development technology, the extraction and utilization of natural active ingredients have become a research hotspot in many 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 activity 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, anti-tumor peptides, etc.), antioxidant factors and polysaccharide compounds. These substances show great potential in anti-infection, anti-inflammation, and promoting tissue regeneration.

[0003] Among amphibians, Caudata species such as salamanders and ambystoma have attracted widespread attention for their excellent regenerative abilities and rich skin secretion characteristics. Studies have shown that the skin of Caudata 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 medicines and biological products. The antimicrobial peptides secreted from the skin of Caudata species not only have broad-spectrum antibacterial properties, but also have a significant inhibitory effect on a variety of drug-resistant strains, providing a potential solution to the problem of antibiotic abuse. In addition, the regenerative factors in the skin of Caudata species are of great 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 active ingredients in the skin of Caudata species such as salamanders and ambystoma started late, and the exploration of their applications is still in its infancy, and the application range of their skin extracts is also greatly limited.

[0004] Therefore, it is of great significance to promote the development of this field to solve the problems of the current lack of research on skin extracts of Caudata species such as salamanders and ambystoma, limited application scope, and serious lack of application fields, explore efficient, stable and sustainable active ingredient extraction technology, provide a wider range of application fields, and fill the gap in the practical application of skin extracts of amphibian Caudata species. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an Ambystoma salamander skin extract and a preparation method and application thereof, aiming to solve the problem that the current amphibian Caudata skin extract has little research, limited application scope, and serious lack of application fields.

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

[0007] The skin extract of the amniotic fluid provided by the present invention is rich in a variety of active ingredients, among which the collagen (COL) family and the fibrin (FCN) family can improve the skin moisture content and reduce the transepidermal water loss of the skin, improve skin wrinkles and elasticity, and improve the immune protein of the skin collagen fiber structure complement system; C1Q complement protein (Complement Component 1q) plays a key role in neuronal function and aging; Fibronectin (FN1 or FN) has the function of a growth factor, which 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 extracellular matrix of the skin, it can firmly fix collagen on the 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 strengthen the skin's defense; cytochrome b5 reductase 3 (cytochrome b5 Overexpression of cytochrome b5 reductase 3 (CYB5R3) activates respiratory metabolism and can extend lifespan; while cytochrome b5 reductase 3, nicotinamide phosphoribosyltransferase (NAMPT), and mitochondrial aldehyde dehydrogenase 2 (ALDH2) have a promoting effect on metabolism and play an important role in cell aging and immune cell function regulation. The above-mentioned rich bioactive ingredients complement and promote each other, making the amniotic fluid skin extract have a wide range of application prospects in drug 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 Ambystoma salamander 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 C1qSubcomponent C), C1QB (Complement C1q Subcomponent B), C1QA (Complement C1qSubcomponent A), or C1QTNF3 (Clq and TNF related 3).

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

[0012] Mexican axolotl (scientific name: Ambystoma mexicanum ) is an amphibian of the family Ambystoma and genus Ambystoma, also known as the six-horned salamander, the six-horned dinosaur, and the American axolotl. The wild Mexican axolotl is dark brown with black spots. It has a wide head, small eyes, and small limbs and feet. The body length is usually 20 to 30 cm and the weight is 60 to 110 grams. The skin is smooth and scaleless, without a tympanic cavity or eardrum. It has obvious external gills and a fin-like tail that is feathery. The dorsal fin extends from the back of the head to the end of the tail, and the pelvic fin extends from the middle of the two hind limbs to the end of the tail, which is conducive to swimming in the water. The Mexican axolotl has become a research hotspot because of its extremely strong regenerative ability.

[0013] At present, the research on the skin of amphibian Caudata species is still in its early stages, and the extraction methods of its active ingredients are mostly borrowed from other amphibians (such as frogs and toads). In the existing technology, the following methods and technical routes are usually used for the extraction and separation of active extracts from animal skin: (1) Solvent extraction: This is the most traditional and widely used method. It uses water, ethanol or other organic solvents as extraction media. By immersing the skin tissue in the solvent, the active ingredients are released by molecular solubility. The advantages of this method are simple operation and low cost, but it is not friendly to heat-sensitive substances or components with easily damaged structures. (2) Enzymatic extraction: By adding specific enzymes (such as proteases and cellulases), the skin tissue is selectively degraded to release proteins, peptides 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 is a problem of difficulty 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, so that the active ingredients in the cells can be quickly released into the extraction medium. This method has a short operation time and high extraction efficiency, and is particularly suitable for the initial separation step. However, in practical applications, the intensity and treatment time of ultrasound need to be strictly controlled, otherwise it may cause degradation of the active substance. (4) Mechanical disruption and centrifugation: Mechanical disruption technology uses a homogenizer or tissue grinder to physically disrupt the skin sample to expose and release the cell contents, and then combines high-speed centrifugation technology to separate the precipitate and the supernatant. This method is flexible and suitable for small-scale extraction, but a large amount of impurities may be mixed in the process and require further purification. (5) Membrane separation and chromatography technology: For further purification of crude extracts, membrane separation technology and chromatography technology (such as high-performance liquid chromatography HPLC) are widely used. Membrane separation removes large molecular 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 implement on a large scale. (6) Freeze drying and stabilization treatment: In order to better preserve the active ingredients, some existing technologies will freeze-dry the product after extraction to remove moisture at low temperatures, thereby improving the stability of the extract. The use of stabilizers (such as antioxidants) can further extend the shelf life of the extract, especially the activity of heat-sensitive substances.

[0014] The above methods provide basic support for the research of active extracts from amphibian skin. However, due to the particularity of the skin of Caudata species, most of the existing technologies are general extraction processes, and no optimization scheme has been formed specifically for the specific components of Caudata skin. Not only that, the methods of solvent extraction, enzymatic hydrolysis and physical separation still have many shortcomings in extraction efficiency, component stability and protection of target active substances, resulting in low extraction efficiency and limited yield of active ingredients. In addition, the extraction process also has problems such as easy protein degradation, impurity mixing and unsuitability for large-scale production, which greatly limits the practical application scope of active extracts from the skin of salamanders, amphibian and other species. To this end, it is particularly important to develop a set of efficient extraction and separation schemes tailored to the skin characteristics of Caudata species, improve the separation and purification efficiency of active ingredients, protect the stability of target active substances, optimize the extraction process and operational adaptability, and realize large-scale and industrial production.

[0015] Based on this, in order to solve the problems of protein degradation in the skin extraction process of amphibian and urodendron species such as salamanders and amphibian newts, protein structure changes caused by enzymatic extraction, excessive impurity content in the extraction process, and extract properties that are not suitable for human application, the second aspect of the present invention provides a method for preparing the amphibian newt skin extract, comprising the steps of: S100, sampling the skin of the Ambystoma salamander; S200, performing liquid nitrogen quick freezing and freeze-thawing on the obtained skin samples; S300, mechanically crushing the frozen-thawed sample; S400, centrifugally filtering the crude extract of the mechanically crushed sample; S500, subjecting the sample extract after centrifugal filtration to ultrasonic disruption; S600, sterilizing and filtering the secondary extract after ultrasonic disruption to obtain an Ambystoma salamander skin extract.

[0016] According to the preparation method of the second aspect of the present invention, there are at least the following beneficial effects: the present invention develops an efficient, stable and sustainable optimized extraction scheme for the skin characteristics of amphibians and Caudata species, integrates the whole process technology from sample processing to extraction and separation, and ensures the efficient separation and activity retention of the extract. The method for extracting skin tissue protein of Caudata species provided by the present invention includes skin sampling, sample freezing and thawing, mechanical crushing, centrifugal filtration, ultrasonic crushing, sterilizing filtration and other steps, and can extract a large amount of protein from species such as salamanders and amphibian newts: first, liquid nitrogen quick freezing treatment is used when processing skin samples to achieve rapid freezing of samples to inhibit the growth of microorganisms and the activity of enzymes, thereby effectively avoiding protein degradation; mechanical crushing is used to replace enzymatic extraction method to maintain the integrity of protein structure, while significantly increasing the protein content, which is suitable for large-scale operation requirements; ultrasonic crushing is used to further remove impurities, and sterilizing filtration is performed to thoroughly remove impurities, thereby improving the purity of the extract; at the same time, the pH value of the extract is adjusted from weak alkalinity to weak acidity during the extraction process to improve the compatibility and adaptability to human skin. Finally, the optimized extraction and separation method proposed in the present invention, targeting the tissue characteristics of the skin of Caudata species, gradually extracts the active ingredients in the skin of Caudata such as salamanders and ambystoma salamanders under low temperature conditions throughout the process by performing rapid freeze-thaw treatment on the samples and combining mechanical crushing, ultrasound-assisted separation and multiple filtration steps, avoiding the negative effects of enzymes, organic solvents or high temperatures on the activity of the target products, and obtaining a variety of complex proteins; 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 needs of human skin, and it is sterile and can be directly used as a product raw material; the above-mentioned extraction technology is green, environmentally friendly, and sustainable, and can achieve large-scale and industrial production, which can significantly promote the industrial application of extracts from the skin of Caudata such as salamanders and ambystoma salamanders, which 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.

[0017] In some embodiments of the present invention, the preparation method can also be applied to extract skin active ingredients from other amphibian Caudata species.

[0018] Specifically, the preparation method can also be applied to the preparation of salamander skin extract, comprising the steps of: S10, sampling the skin of salamanders; S20, performing liquid nitrogen quick freezing and freeze-thawing on the obtained skin samples; S30, mechanically crushing the frozen-thawed sample; S40, centrifugally filtering the crude extract of the mechanically crushed sample; S50, subjecting the sample extract after centrifugal filtration to ultrasonic disruption; S60, sterilizing and filtering the secondary extract after ultrasonic disruption to obtain a salamander skin extract.

[0019] In some embodiments of the present invention, after sampling the skin of the amphibian, the obtained skin sample is stored in a buffer containing a protease inhibitor.

[0020] In some embodiments of the present invention, the pH value of the buffer is 5.5-6.

[0021] Specifically, the buffer solution formula is: phosphate buffer containing 1× protease inhibitor, pH 6.0. Compared with other extraction methods, the present invention adds protease inhibitors to prevent protein degradation and obtain high-content protein.

[0022] In some embodiments of the present invention, step S100 specifically includes: S101. Anesthetize and kill the amphibian; S102. Skin removal: The killed amniotic fluid was laid flat on a table, and a scalpel was used to vertically cut along both sides of the spine of the back, and horizontally cut along a circle around the neck, and then the skin was peeled off with tweezers, and the peeled skin of the amniotic fluid was weighed; S103, cleaning: immersing the skin of the amphibian in phosphate buffer for cleaning, and cleaning at least 3 times; S104, cutting into pieces: using absorbent paper to remove surface moisture from the cleaned skin of the amphibian, placing it in a culture dish, and using scissors to cut it into small pieces of 0.3×0.3 cm in size; S105. Storage: Transfer a small piece of amniotic fluid skin to a clean centrifuge tube and add a buffer solution equal in weight to the amniotic fluid skin.

[0023] In step S104, cutting into small pieces is to ensure that the skin of the amniotic fluid is fully frozen by liquid nitrogen.

[0024] In conventional methods, the final extract is weakly alkaline, and its compatibility with human skin is poor when used directly, making it difficult to be used as a cosmetic raw material. The present invention adjusts the pH value of the extract from weak alkalinity to weak acidity by adjusting the pH value of the phosphate buffer, 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 the extraction is completed, the present invention adjusts the pH value before the extraction, which can prevent damage to the extract during the adjustment process.

[0025] In some embodiments of the present invention, step S200 specifically includes: S201, liquid nitrogen quick freezing: immersing the centrifuge tube containing the small piece of Ambystoma salamander skin into liquid nitrogen until the small piece of Ambystoma salamander skin and the buffer solution are completely frozen; S202, freeze-thaw: Place the sample after quick freezing with liquid nitrogen in a -80℃ refrigerator for 24 hours, take it out and place it at room temperature until it is completely thawed; then place it in a -80℃ refrigerator again, freeze it for 24 hours, take it out and place it at room temperature until it is completely thawed; then place it in a -80℃ refrigerator again, freeze it for 24 hours, take it out and place it at room temperature until it is completely thawed, and then proceed with the extraction process.

[0026] During the skin extraction process, in order to prevent protein degradation during sample pretreatment, the present invention uses phosphate buffer and adds 1× concentration of protease inhibitors, combined with immersion liquid nitrogen quick freezing treatment, to achieve rapid freezing of samples to inhibit the growth of microorganisms and enzyme activity, thereby effectively avoiding protein degradation and ensuring that protein activity is preserved.

[0027] In some embodiments of the present invention, step S300 specifically includes: S301, transfer the completely thawed sample to a clean centrifuge tube and use a handheld high-speed homogenizer for mechanical disruption. The entire disruption process is performed on ice; S302, place the centrifuge tube on ice and wait for centrifugation.

[0028] Specifically, the sample should not exceed 1 / 2 of the volume of the centrifuge tube to prevent liquid splashing during mechanical crushing.

[0029] Specifically, the mechanical disruption parameters were as follows: disruption for 15 minutes, stopping every 5 minutes, removing the tendons wrapped around the probe and discarding them, and the entire disruption process was carried out on ice to control the temperature so that the protein would not denature.

[0030] Most conventional methods use biological enzyme dissociation, and the skin sample is prone to changes in protein structure during the enzymatic extraction process; and this method takes a long time and is not suitable for large-scale operations. The solution adopted by the present invention is: to replace the enzymatic extraction method with a mechanical crushing method to maintain the integrity of the protein structure, while significantly increasing the protein content, which is suitable for large-scale operation needs (50 to 100 salamander or amphibian skin can be extracted at a time); moreover, the inactivation of biological enzymes may require a high temperature of 60°C, which will affect the protein structure. The present invention uses mechanical crushing and operates on ice throughout the process to control the temperature so that the protein does not denature.

[0031] In some embodiments of the present invention, step S400 specifically includes: S401, initial centrifugation: 4°C, 4000 g, centrifugation for 10 min; S402, transfer the clear liquid: use a Pasteur pipette to pass through the upper layer of impurities and foam, take the middle layer of clear liquid, and transfer it to a clean centrifuge tube; S403, centrifuge again: 4°C, 4000g, centrifuge for 10 minutes; S404, filtration: take the supernatant, filter it using a 40 μm cell sieve, and collect the filtrate into a clean centrifuge tube; S405, filtration again: Filter the collected filtrate again using a 10 μm filter, and collect the liquid into a clean centrifuge tube for ultrasonic crushing.

[0032] The purpose of centrifugation of the crude extract is to remove larger impurities. Two centrifugations can simply and quickly remove impurities without the need for salting out or ultrafiltration. Two filtrations can fully filter out obvious impurities visible to the naked eye, waiting for further ultrasonic crushing to remove tiny impurities.

[0033] In some embodiments of the present invention, step S500 specifically includes: S501, transfer the liquid into a beaker, and place the beaker on ice; S502, ultrasonic fragmentation: set the fragmentation power to 1%, ultrasonic on time to 4.0s, ultrasonic off time to 6.0s, working time to 30 minutes, control the temperature below 35°C, immerse the probe to 0.5cm from the bottom of the beaker, and perform the entire ultrasonic process on ice; S503. After the ultrasonic disruption is completed, transfer the liquid to a clean centrifuge tube.

[0034] During ultrasonic disruption, the liquid in the beaker should not exceed 1 / 2 of the beaker volume (for example, for a 50mL beaker, the liquid volume should be controlled at 20~25mL).

[0035] The crude extract contains a lot of impurities, which affects subsequent applications. The present invention uses ultrasonic crushing to further remove impurities, and the whole process is operated on ice to ensure that the protein will not be denatured by high temperature. After crushing, it is filtered twice with a 0.22 μm filter to completely remove impurities, thereby improving the purity of the extract.

[0036] In some embodiments of the present invention, the secondary extract after ultrasonic disruption is subjected to double sterilization filtration, and the filter membrane pore size used in the double sterilization filtration is 0.22 μm.

[0037] In some implementations of the present invention, step S600 specifically includes: S601, centrifuge the ultrasonically disrupted liquid at 4°C, 4000 g for 10 minutes, and transfer the supernatant to a clean centrifuge tube; S602, vacuum filtration: Use vacuum filter / 0.22µm pore size / PES filter membrane for filtration, connect the filter bottle to the vacuum pump, pour the supernatant into the filter bottle, start the vacuum pump, and filter; S603, Filtration: Use a 0.22µm pore size / CA membrane pinhole filter to filter again, connect the syringe to the syringe filter, pour the liquid into the syringe, push the filter, and collect the filtered liquid into a clean centrifuge tube; S604. Storage: The collected Ambystoma salamander skin extract is stored at -80°C.

[0038] The invention adopts double sterilization filtration, and the pore size of the filter membrane is 0.22 μm. The obtained Ambystoma salamander extract is free of impurities and sterile, thereby ensuring the availability of the extract.

[0039] In some embodiments of the present invention, after step S600, step S700 is further included: BCA protein assay.

[0040] Specifically, step S700 includes: S701: Dilution: Take the skin extract of Ambystoma salamander and dilute it 10 times, 20 times, and 30 times; S702: protein concentration was determined using a BCA protein concentration assay kit; S703: Calculate the protein concentrations of 10-fold, 20-fold, and 30-fold dilutions and take the average value.

[0041] In summary, the method for extracting the skin of the amphibian provided by the present invention uses a phosphate buffer and adds a 1× concentration of protease inhibitor to prevent protein degradation during sample processing, and achieves a rapid freezing effect by immersion in liquid nitrogen quick freezing, thereby inhibiting the growth of microorganisms and the activity of enzymes, and ensuring that the activity of proteins is preserved; in view of the problem that the traditional enzymatic extraction method is prone to cause changes in protein structure and is not suitable for large-scale operations, a mechanical crushing method is used instead, and the protein content is kept high and the structure is intact by physical crushing means, while simplifying the process flow and improving industrial operability; in order to further remove impurities in the extract, the present invention combines ultrasonic treatment technology, and operates on ice throughout the process to ensure that the protein is not denatured due to high temperature. The extract after ultrasonic treatment is filtered twice with a 0.22μm filter to remove fine impurities and improve the purity of the extract; at the same time, in order to improve the chemical properties of the extract to adapt to human skin applications, the present invention adjusts the pH value of the phosphate buffer from weak alkalinity to weak acidity, so that the final product is 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 amphibians such as salamanders.

[0042] Through the above-mentioned innovative process, the present invention realizes a stable preparation method for skin extracts of Caudata species. The entire process adopts physical extraction methods to avoid the damage to the quality and activity of the extract by enzymatic hydrolysis or chemical reagents. At the same time, the structural characteristics of the skin of Caudata such as salamanders and ambystoma are fully considered. Through precise multi-step processing to remove impurities and optimize product properties, a high-purity, high-activity and sterile skin extract is successfully obtained. The process of the present invention has undergone a number of functional verification experiments, which have proved that it has high stability and applicability, and has shown a wide range of application potential.

[0043] An embodiment of the third 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 anti-aging, wherein the application is for non-diagnostic or therapeutic purposes.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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: (a1) developing and / or preparing skin anti-aging products; (a2) developing and / or preparing skin care products; (a3) developing and / or preparing drugs for treating aging-related skin diseases; (a4) Developing and / or preparing drugs for repairing skin damage; (a5) constructing a skin aging model; (a6) constructing a skin damage repair model; (a7) Developing and / or preparing an elastase inhibitor, a hyaluronidase inhibitor, a 5α-reductase inhibitor, or a free radical scavenger.

[0048] In some embodiments of the present invention, the application includes developing and / or preparing skin anti-aging products or skin care products. The Ambystoma skin extract of the present invention can effectively match the needs of human skin due to its sterile, high purity and weak acidity, 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, essences, 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 removal, improvement of sensitive skin and repair of skin barrier.

[0049] In some embodiments of the present invention, the application includes the development and / or preparation of drugs for treating aging-related skin diseases or skin damage repair drugs. The Ambystoma skin extract of the present invention contains rich bioactive ingredients, including polypeptides and proteins with antibacterial, anti-inflammatory, antioxidant and other functions. In the field of medicine, these ingredients 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 way for the development of drugs for burns, postoperative wounds and chronic skin ulcers by promoting collagen production, inhibiting inflammatory reactions and microbial infections. At the same time, the extract has potential value in the development of anti-aging drugs, and can slow down the cell aging process and enhance cell activity.

[0050] In some embodiments of the present invention, the application includes developing and / or preparing functional foods or health products.

[0051] Due to the natural origin of the Ambystoma skin extract and its biological activity, the product of the present invention has the potential to become a raw material for functional foods and health products. Its antioxidant properties help reduce the damage of free radicals to the body, thereby improving 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 protective mechanisms.

[0052] In some embodiments of the invention, the use comprises developing biomaterials.

[0053] 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.

[0054] In some embodiments of the present invention, the application includes constructing a drug screening platform.

[0055] 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.

[0056] The above applications provide a solid foundation for technological progress in multiple fields and demonstrate broad market value and social benefits.

[0057] 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.

[0058] In some embodiments of the present invention, the product comprises a medicine, a pharmaceutical composition, a skin care product, or a cosmetic.

[0059] 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.

[0060] In some embodiments of the present invention, the medicine, pharmaceutical composition, skin care product, or cosmetic further comprises a pharmaceutically or skin care product acceptable carrier.

[0061] In some embodiments of the present invention, the skin care products or cosmetics include anti-wrinkle products, firming products, moisturizing products, soothing products, oil control products, and anti-photoaging products.

[0062] In some embodiments of the present invention, the categories of the skin care products or cosmetics include essence, lotion, cream, and mask.

[0063] In some embodiments of the present invention, the ingredients of the skin care product or cosmetic further include at least one of glycerin, hyaluronic acid, propylene glycol, or butylene glycol.

[0064] In some embodiments of the present invention, the product comprises at least one of the following (b1) to (b8): (b1) Skin anti-aging products; (b2) Skin care products; (b3) Drugs for treating skin diseases associated with aging; (b4) Skin damage repair drugs; (b5) elastase inhibitors; (b6) hyaluronidase inhibitors; (b7) 5α-reductase inhibitors; (b8) free radical scavengers; The active ingredient of the product includes the above-mentioned Ambystoma salamander skin extract or the Ambystoma salamander skin extract obtained by the above-mentioned preparation method.

[0065] In some embodiments of the present invention, the product is an external preparation prepared by using the amniotic fluid skin extract as an active ingredient and adding excipients or auxiliary ingredients acceptable in medicine or cosmetics.

[0066] In some embodiments of the present invention, the external preparation comprises a cosmetic or a skin care product.

[0067] In some embodiments of the present invention, the cosmetics or skin care products include anti-wrinkle creams, firming creams, moisturizing lotions or milks, soothing gels or gels, oil-control creams or lotions, and anti-photoaging creams or powders.

[0068] In some embodiments of the present invention, the types of cosmetics or skin care products include water-based agents, emulsions, creams, powders, gels, essences, or facial masks.

[0069] In some embodiments of the present invention, the acceptable excipients include at least one of a diluent, a binder, a wetting agent, a humectant, a thickener, a solvent, an emulsifier, a cosolvent, a preservative, a pH adjuster, an osmotic pressure regulator, a surfactant, and a buffer.

[0070] In some embodiments of the present invention, the auxiliary ingredients include at least one of a whitening agent, an emollient, an anti-acne agent, an ultraviolet light absorber, and a skin conditioning agent.

[0071] In some embodiments of the present invention, the auxiliary ingredients also include at least one of collagen, vitamins, tea polyphenols, and coenzyme Q10.

[0072] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 A schematic diagram of the BCA concentration measurement results provided in an embodiment of the present invention; Figure 2 A schematic diagram of the anti-wrinkle and firming efficacy test results of the Ambystoma salamander skin extract provided in an embodiment of the present invention; Figure 3 A schematic diagram of the moisturizing effect test results of the Ambystoma salamander skin extract provided in an embodiment of the present invention; Figure 4 A schematic diagram of the soothing efficacy test results of the Ambystoma salamander skin extract provided in an embodiment of the present invention; Figure 5 A schematic diagram of the oil control efficacy test results of the Ambystoma salamander skin extract provided in an embodiment of the present invention; Figure 6 A schematic diagram of the anti-photoaging efficacy test results of the Ambystoma salamander skin extract provided in an embodiment of the present invention; Figure 7 Schematic diagram of the evaluation results of the skin damage repair efficacy of the Ambystoma skin extract provided in the embodiment of the present invention: the wound morphology of the control group on the 20th day; Figure 8 Schematic diagram of the evaluation results of the skin damage repair efficacy of the Ambystoma skin extract provided in the embodiments of the present invention: the wound morphology of the experimental group on the 20th day. DETAILED DESCRIPTION

[0074] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0075] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0076] In the description of the present invention, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are real numbers. Unless otherwise specified, each reaction or operation step may be performed in sequence or not. Preferably, the reaction method of the present invention is performed in sequence.

[0077] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0078] Example 1 Preparation of Ambystoma salamander skin extract 1) Purchase 35 white Mexican axolotls with a size of 15-17 cm from a farm with artificial breeding qualifications (Jiutai District Karen Yiwen Ornamental Fish Farm, Group 6, Karen Sunjia Village, Jiutai District, Changchun City, Jilin Province).

[0079] 2) Before sampling, the fish anesthetic 3-aminobenzoic acid ethyl ester methanesulfonate (J&K Technology, 247097) was used to anesthetize the amniotic fluid. The anesthesia scheme was as follows: 1.5 g of anesthetic was added to 1 L of water, and the amniotic fluid was placed in the water. The amniotic fluid was anesthetized for 15 minutes without moving the body. The amniotic fluid was then killed and the skin was taken: the amniotic fluid was laid flat on the table, and a scalpel was used to make vertical cuts along both sides of the spine on the back, and a horizontal cut was made along the neck. The skin was peeled off with tweezers. Finally, about 170 g of amniotic fluid skin was obtained.

[0080] 3) Wash the skin of the amphiprion in phosphate buffered saline three times.

[0081] 4) After cleaning, use absorbent paper to absorb the moisture on the surface of the blunt-stoma salamander skin.

[0082] 5) Place the skin in a 10 cm culture dish and cut it into small pieces of 0.3 × 0.3 cm with scissors (to ensure that the 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 inhibitors at an equal weight.

[0083] 6) Immerse the 15 mL centrifuge tube containing the amphibian skin piece into liquid nitrogen for 5 minutes until the amphibian skin piece and buffer are completely frozen; 7) After quick freezing, place in a -80℃ freezer. After 24 hours of freezing, take out and thaw at room temperature.

[0084] 8) Place the sample back into the -80℃ freezer, freeze for 24 hours, and thaw at room temperature.

[0085] 9) Repeat step 8 once.

[0086] 10) After complete thawing, transfer the sample to a clean 50mL centrifuge tube with the liquid level not exceeding 25mL to prevent liquid splashing during mechanical crushing; use a handheld high-speed homogenizer to crush the sample. The mechanical crushing parameters are: crush for 15 minutes, stop every 5 minutes, remove the meat tendons wrapped around the probe and discard them. The entire crushing process is carried out on ice to control the temperature so that the protein does not denature.

[0087] 11) After complete grinding, centrifuge at 4°C, 4000g for 10 minutes.

[0088] 12) Use a Pasteur pipette to pass through the upper layer of impurities and foam, take the middle layer of clear liquid, transfer it to a clean 50mL centrifuge tube, and centrifuge again at 4°C, 4000g for 10 minutes.

[0089] 13) Transfer the supernatant for filtration, filter using a 40 μm cell sieve, and collect the filtrate into a clean 50 mL centrifuge tube.

[0090] 14) Filter again using a 10 μm filter and collect the liquid into a clean 50 mL centrifuge tube.

[0091] 15) Transfer the filtered salamander skin extract to a 50 mL beaker with a volume of 20-25 mL and place the beaker on ice.

[0092] 16) Set the crushing 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 0.5cm from the bottom of the beaker, and perform the entire ultrasonic process on ice.

[0093] 17) After ultrasonic disruption, transfer the liquid to a clean 50 ml centrifuge tube and centrifuge at 4°C, 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.

[0094] 18) Use Corning 500 mL vacuum filter / 0.22 µm pore size / PES filter membrane for filtration. Connect the filter bottle to the vacuum pump, pour the liquid into the filter bottle, start the vacuum pump, filter, and collect the filtered liquid.

[0095] 19) Filter again using a 0.22 µm pore size / CA membrane needle filter. Connect the syringe to the syringe filter, pour the liquid into the syringe, push the filter, and collect the filtered liquid into a clean 50 mL centrifuge tube to obtain a total of 140 mL of extract liquid.

[0096] 20) Use the BCA Protein Concentration Assay Kit (Enhanced) to determine the BCA concentration.

[0097] Some of the reagents and manufacturers used in this example are shown in Table 1: Table 1

[0098] The results of BCA concentration determination were as follows Figure 1 From Table 2, we can see that the protein concentration of the extract is 18.4 mg / mL, the total volume of the extract is 140 mL, and the total protein content is 2576 μg.

[0099] Table 2

[0100] Example 2 Sterility Test of Ambystoma Skin Extract 1 mL of the amphibian skin extract of Example 1 was taken for sterility testing. The test items were as follows: Klebsiella pneumoniae, Salmonella, Escherichia coli, Bordetella bronchiseptica, Pasteurella multocida, Pasteurella pneumophila, beta-hemolytic Streptococcus, Streptobacillus moniliformis, Corynebacterium rapae, Staphylococcus aureus, and Pseudomonas aeruginosa. The test results were all sterile.

[0101] Example 3 Detection of active substances in the skin extract of Ambystoma salamander Take 10 mL of the skin extract of the amphibian of Example 1 for mass spectrometry detection, and confirm the contained proteins based on the peptide spectrum. The specific method is: the corresponding peptide is broken at the peptide bond position by ETD (electron transfer dissociation), which will produce corresponding b daughter ions and y daughter ions. Each b / y daughter ion has a corresponding mass-to-charge ratio (mass divided by charge) and a corresponding signal intensity, and a theoretical mass spectrum is generated (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, thereby inferring the peptide sequence corresponding to the actual spectrum.

[0102] After analysis, it was found that the skin extract of Ambystoma salamander contains a variety of active ingredients, including: a variety of 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 proteins in the fibrin family, such as FCN1 protein, FCN3 protein, FCN2 protein, etc.; various proteins 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 acetaldehyde dehydrogenase 2 (ALDH2). (The protein spectrum test results are not displayed) Example 4 Efficacy testing and safety evaluation of Ambystoma salamander skin extract 40 mL of the amphibian skin extract stock solution of Example 1 was taken for efficacy and safety evaluation, and the test items included anti-wrinkle, wrinkle tightening, moisturizing, soothing, oil control, and anti-photoaging efficacy evaluation. The test basis includes laboratory methods LC-WI-HZ-176 Elastase Inhibition Test Operation Instructions, LC-WI-HZ-180 Moisturizing Efficacy Test Experimental Instructions, LC-WI-HZ-185 Hyaluronidase Inhibition Experimental Operation Instructions, LC-WI-HZ-178 5α-reductase Inhibition Experimental Operation Instructions, and LC-WI-HZ-174 Free Radical (DPPH) Scavenging Experimental Operation Instructions.

[0103] 1. Anti-wrinkle and firming efficacy test results Elastic fibers are composed of elastin and microfibrils, distributed in the dermis and subcutaneous tissue, making the skin elastic. However, due to the effects of some environmental factors such as ultraviolet radiation, pressure, pollution, etc., the production of elastase in the body will be promoted. Elastase is a member of the chymosin family, which degrades elastin, causing the loss of connecting tissue in the epidermis, thereby leading to loose skin and the generation of wrinkles. Therefore, inhibiting the generation of elastase can block or prevent the occurrence of loose skin and wrinkles, and achieve anti-wrinkle and firming effects. In this embodiment, the results of elastase inhibition rate are compared by using a sample of amniotic stomata skin extract and a blank control group. If the inhibition rate of the sample of amniotic stomata skin extract is better than (higher than) the inhibition rate of the blank control group and the statistical difference P value is <0.05, it can be considered that the sample has certain anti-wrinkle and firming effects.

[0104] The steps are as follows: pre-treat each group of samples to prepare a mixed solution; incubate in an incubator at 25°C for 15 minutes; then add a certain amount of elastase and continue incubating for 15 minutes; use an enzyme-labeled analyzer to test the OD value of the elastase inhibition rate at a wavelength of 410nm; calculate the inhibition rate and statistically calculate the difference P value.

[0105] The results are as follows Figure 2 And as shown in Table 3.

[0106] Table 3

[0107] The results show that the inhibition rate of the Ambystoma skin extract sample is better than (higher than) the inhibition rate of the blank control group and the statistical difference P value is <0.05, indicating that the Ambystoma skin extract sample has firming and anti-wrinkle effects.

[0108] 2. Moisturizing efficacy test results The water in the stratum corneum of the skin plays an important role in maintaining skin health, preventing skin aging and moisturizing. The reason for dry skin is insufficient water in the stratum corneum, and the water is often combined with the stratum corneum in the form of bound water. In this embodiment, the water weight change of the treated Ambystoma salamander skin extract test sample is continuously monitored to draw a water change rate Δm-time change curve of the test sample. The moisturizing ability of the test sample is observed according to the trend of the water change rate Δm curve, and then the moisturizing effect of the test sample on the skin is evaluated.

[0109] The steps are: pre-treating the sample, placing the sample and the positive control (glycerol is used in this embodiment) in a constant temperature and humidity chamber under certain temperature and humidity conditions, weighing them regularly using an electronic balance, recording data, calculating the moisture change rate Δm, and making a curve.

[0110] The results are as follows Figure 3 And as shown in Table 4.

[0111] Table 4 Test results of moisture change rate Δm

[0112] The results show that the moisture change rate Δm curve of the Ambystoma salamander 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.

[0113] 3. Soothing effect test results 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 inflammatory reactions, thereby alleviating the discomfort caused by skin inflammation. Macromolecular hyaluronic acid will degrade under the action of hyaluronidase, resulting in a decrease in the content of macromolecular hyaluronic acid. Decomposition produces a degradation product, N-acetylglucosamine. Determining the content of N-acetylglucosamine in the reaction system can indirectly reflect the activity of hyaluronidase. This embodiment determines the inhibition rate of the test sample on hyaluronidase by determining the content of N-acetylglucosamine in the reaction system. If the inhibition rate of the test sample is better than (higher than) the inhibition rate of the blank control group and the statistical difference P value is <0.05, it can be considered that the test sample has a certain soothing effect.

[0114] The steps are as follows: pretreat the test samples, prepare sample tubes with different dilutions, and make parallel blank groups and positive controls; add reactants to each group and mix them well, then place them in a constant temperature water bath at 37°C for 40 minutes; add a pH adjuster, bathe in boiling water for 15 minutes, bathe in ice for 10 minutes, and then cool to room temperature; use an enzyme-labeled analyzer to measure the OD value at a wavelength of 530nm, and calculate the hyaluronidase activity inhibition rate and the statistical difference P value.

[0115] The results are as follows Figure 4 And as shown in Table 5.

[0116] Table 5

[0117] The results show that the inhibition rate of the Ambystoma skin extract sample is better than (higher than) the inhibition rate of the blank control group and the statistical difference P value is <0.05, indicating that the Ambystoma skin extract sample has a soothing effect.

[0118] 4. Oil control efficacy test results 5α-reductase (5α-reductase) is a membrane protease that depends on reduced coenzyme II (NADPH). It is an important male hormone metabolic enzyme in the skin. It can irreversibly convert testosterone (Testosterone, T) into dihydrotestosterone (Dihydrotestosterone, DHT). DHT is the most active male hormone and can induce excessive secretion of oil from sebaceous glands. Reducing the level of DHT by inhibiting the activity of 5α-reductase can effectively alleviate the excessive secretion of oil from sebaceous glands. In this embodiment, the results of the 5α-reductase inhibition rate test are compared between the test sample and the blank control group. If the inhibition rate of the test sample is better than (higher than) the inhibition rate of the blank control group and the statistical difference P value is <0.05, it can be considered that the test sample has a certain oil control effect.

[0119] The steps are as follows: pre-treat 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, continue the reaction, and place it in an incubator at 37°C for 30 minutes; first add the color developer to each well, develop the color in an incubator at 37°C in the dark, add the stop solution, and stop the reaction; measure the absorbance of each well on the machine test, record the data, and calculate the inhibition rate.

[0120] The results are as follows Figure 5 and as shown in Table 6.

[0121] Table 6

[0122] The results show that the inhibition rate of the Ambystoma skin extract sample is better than (higher than) the inhibition rate of the blank control group and the statistical difference P value is <0.05, indicating that the Ambystoma skin extract sample has an oil-control effect.

[0123] 5. Anti-photoaging efficacy test results Ultraviolet radiation in sunlight can cause photoaging of human skin tissue, one of the characteristics of which is aging and wrinkles on the skin of the light-exposed part. There are many mechanisms that cause skin photoaging, one of which is that light radiation can lead to the generation of free radicals. Free radicals have extremely strong oxidizing ability. They attack any biological molecules through oxidation reactions, and undergo various oxidation reactions with macromolecules such as lipids, sugars, proteins, and deoxyribonucleic acid, causing oxidative damage such as denaturation, cross-linking, and breakage, which in turn leads to the destruction of cell structure and function, as well as damage to body tissues and lesions of organs, which manifest as photoaging on the skin. When the test sample is in the presence of a free radical scavenger, the light absorption of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) reagent (a stable long-lived free radical) is weakened. This can evaluate the ability of the test sample to scavenge free radicals, that is, the size of the antioxidant activity. In this embodiment, a DPPH free radical scavenging rate test is performed 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 is <0.05, it can be considered that the test sample has a certain anti-photoaging effect.

[0124] The steps are as follows: pretreat the test samples, prepare sample tubes of different dilutions, and make parallel blank groups and positive controls; add free radical DPPH, prepare multiple tubes of samples, shake well, and set aside; after standing at room temperature for 5 minutes, measure the absorbance value at a wavelength of 517nm using an enzyme-labeled analyzer; record the data, and calculate the clearance rate and statistical difference P value before and after adding the test substance.

[0125] The results are as follows Figure 6 And as shown in Table 7.

[0126] Table 7

[0127] The results show that the clearance rate of the Ambystoma skin extract sample is better than (higher than) the clearance rate of the blank control group and the statistical difference P value is <0.05, indicating that the Ambystoma skin extract sample has anti-photoaging effect.

[0128] Example 5 Evaluation of the skin damage repair efficacy of Ambystoma salamander skin extract This example verifies the skin damage repairing efficacy of the Ambystoma salamander skin extract through a SD rat scald model and intervention experiments.

[0129] 1. Use 8-week-old SD rats and divide them into two groups (control group and experimental group) randomly and evenly.

[0130] 2. Preparation The abdomen of the SD rats was shaved to ensure smooth and hairless skin; a depilatory cream was used to further depilate the shaved area for 9 minutes; after depilation, the depilated area was cleaned with saline and dried for later use.

[0131] 3. Treatment of burns Use a metal ironing table with a diameter of 1.5 cm, adjust the temperature of the ironing table to 200℃, and scald the lower abdomen of the SD rats on both sides once, and each scalding lasts for 12 seconds; after the scalding treatment, observe the scalded area in time and clean the wound surface.

[0132] 4. Group processing Control group: The mask cloth was soaked in 300 μL PBS (phosphate buffered saline), applied to the burn wound and then bandaged aseptically.

[0133] Experimental group: 300 μL of the Ambystoma salamander extract obtained in Example 1 was evenly applied to the burn wound, and a facial mask was applied and sterile bandage was performed.

[0134] 5. Dressing change and observation Week 1: Change medication and dressings daily, and observe and record wound healing.

[0135] Week 2 to Day 20: Change medication and dressing every 3 days, and continue to record wound healing progress, including wound contraction rate, healing area and other indicators.

[0136] 6. Analysis results After the experiment ended on the 20th day, the morphological and histological analyses of the wounds in each group were performed, and ImageJ software was used to compare the differences in wound healing effects between the control group and the experimental group.

[0137] On day 20, the control group ( Figure 7 ) and experimental group ( Figure 8 ) The wound morphology is as follows Figure 7-8 The wound area statistics are shown in Table 8. Table 8

[0138] The results show that the skin damage repair effect of the Ambystoma skin extract sample is better than that of 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 the 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.

[0139] The above is a specific description of the preferred implementation of the present application, but the present application is not limited to the above-mentioned implementation mode. Technical personnel familiar with the field can also make various equivalent deformations or substitutions without violating the spirit of the present application. These equivalent deformations or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. A skin extract of Ambystoma salamander, characterized in that The active ingredients of the Ambystoma salamander skin extract include one or more of the collagen family, one or more of the fibrin family, one or more of the C1Q complement protein family, fibronectin, cytochrome b5 reductase 3, nicotinamide phosphoribosyltransferase, or mitochondrial acetaldehyde dehydrogenase 2.

2. The Ambystoma salamander skin extract according to claim 1, characterized in that The collagen family includes COL1A1, COL1A2, COL3A1, COL4A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL6A6, COL11A1, COL12A1, COL17A1, COL18A1, COL28A1, or COLGALT1; and / or, the ficolin family includes FCN1, FCN3, or FCN2; And / or, the C1Q complement protein family includes C1QC, C1QB, C1QA, or C1QTNF3.

3. The Ambystoma salamander skin extract according to claim 1, characterized in that The axolotls include the Mexican axolotl.

4. A method for preparing the Ambystoma salamander skin extract according to any one of claims 1 to 3, characterized in that: Includes steps: Sampling of the skin of the Ambystoma; The obtained skin samples were snap-frozen in liquid nitrogen and freeze-thawed; The frozen-thawed samples were mechanically disrupted; The crude extract of the mechanically crushed sample is centrifuged and filtered; The sample extract after centrifugal filtration was subjected to ultrasonic disruption; The secondary extract after ultrasonic disruption was sterilized and filtered to obtain the Ambystoma salamander skin extract.

5. The preparation method according to claim 4, characterized in that: After sampling the skin of Ambystoma , the obtained skin samples were stored in a buffer containing protease inhibitors; The pH value of the buffer solution is 5.5-6.

6. The preparation method according to claim 4, characterized in that: The secondary extract after ultrasonic disruption was double sterilized and filtered, and the filter membrane pore size used in the double sterilized filtration was 0.22 μm.

7. Use of the Ambystoma skin extract according to any one of claims 1 to 3 or the Ambystoma skin extract obtained by the preparation method according to any one of claims 4 to 6 in skin anti-aging.

8. The use according to claim 7, characterized in that: The anti-aging includes at least one of anti-wrinkle, firming, moisturizing, soothing, oil control, or anti-photoaging.

9. Use of the Ambystoma skin extract according to any one of claims 1 to 3 or the Ambystoma skin extract obtained by the preparation method according to any one of claims 4 to 6 in repairing skin damage.

10. Use of the Ambystoma salamander skin extract according to any one of claims 1 to 3 or the Ambystoma salamander skin extract obtained by the preparation method according to any one of claims 4 to 6 in at least one of (a1) to (a7): (a1) developing and / or preparing skin anti-aging products; (a2) developing and / or preparing skin care products; (a3) developing and / or preparing drugs for treating aging-related skin diseases; (a4) Developing and / or preparing drugs for repairing skin damage; (a5) constructing a skin aging model; (a6) constructing a skin damage repair model; (a7) Developing and / or preparing an elastase inhibitor, a hyaluronidase inhibitor, a 5α-reductase inhibitor, or a free radical scavenger.

11. A product comprising the Ambystoma salamander skin extract according to any one of claims 1 to 3 or the Ambystoma salamander skin extract obtained by the preparation method according to any one of claims 4 to 6.

12. The product according to claim 11, characterized in that The product includes a medicine, a pharmaceutical composition, a skin care product, or a cosmetic.

13. The product according to claim 12, characterized in that The medicine, pharmaceutical composition, skin care product, or cosmetic further comprises a pharmaceutically or skin care product acceptable carrier.

14. The product according to claim 12, characterized in that The skin care products or cosmetics include anti-wrinkle products, firming products, moisturizing products, soothing products, oil control products, and anti-photoaging products; and / or, the categories of the skin care products or cosmetics include essence, lotion, cream, and mask; And / or, the ingredients of the skin care product or cosmetics further include at least one of glycerin, hyaluronic acid, propylene glycol, or butylene glycol.

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