A biomaterial with hydrophilicity and transdermal absorption promoting property, and a preparation method and application thereof

Abstract

The application discloses a biomaterial with hydrophilicity and transdermal absorption promotion and a preparation method and application thereof, and comprises the following components in mass percentage: 95% of swim bladder extract and 5% of transdermal absorption promotion polypeptide, wherein the swim bladder extract is a hydrophilic substance containing collagen, elastin and glycosaminoglycan and is obtained by extracting swim bladder as raw material; and the amino acid sequence of the transdermal absorption promotion polypeptide is GGPAAVSSQ or AAVPPLTTC. The swim bladder extract rich in collagen, elastin and glycosaminoglycan is used as a hydrophilic substance, and is compounded with the transdermal absorption promotion polypeptide with a specific amino acid sequence, so that the double functions of hydrophilicity and transdermal absorption promotion are obtained, and the biomaterial is better applied to the field of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterial manufacturing, and in particular, is a biomaterial with hydrophilicity and promoting transdermal absorption, and a preparation method and application thereof. BACKGROUND

[0002] Fish swim bladder, commonly known as fish bubble, is a water depth regulator when fish swims, and also a "life buoy" when fish swims, which can adjust the specific gravity of fish body by inflation and deflation. In the processing of fish, a large amount of fish swim bladder is discarded as waste. Fish swim bladder has high moisture retention and low immunological activity, and is widely used in high value-added fields such as functional food, cosmetics, medicine, biological materials (Reference: Research Progress of Fish Swim Bladder Collagen Protein Active Peptide. Journal of Jiamusi Vocational College, 2016(08):431-433.).

[0003] The research results of the paper (Reference: Glycidyl methacrylate-crosslinked fish swim bladder as a novel cardiovascular biomaterial with improved antithrombotic and anticalcification properties. Journal of biomaterials applications, 2022, 36(7), 1188-1200) show that the dry matter of fish swim bladder contains about 70% collagen, 20% elastin and 10% glycosaminoglycan. Among them, glycosaminoglycan is a typical hydrophilic substance, and the content of glycosaminoglycan in fish swim bladder is higher than that in animal skin, blood vessels and other soft tissues.

[0004] A paper (Reference: Study on extraction and properties of collagen from the skin and swim bladder of largemouth bass. Wuhan Polytechnic University, 2012. Master's thesis) reported that the optimal reaction conditions for extracting collagen from the skin of largemouth bass were pH 4, reaction temperature 30°C, substrate concentration 30-40 g / L, enzyme to substrate concentration ratio 1500 u / g, and reaction time 5 h. The optimal reaction conditions for extracting collagen from the swim bladder of largemouth bass were pH 4.8-5, reaction temperature 30°C, substrate concentration 40 g / L, enzyme to substrate concentration ratio 2000 u / g, and reaction time 5 h. The enzyme used in the experiment was papain. The extraction rate of collagen from the skin of largemouth bass was 27.26%, and the extraction rate of collagen from the swim bladder of largemouth bass was 16.73%. Collagen with a smaller molecular weight is more easily absorbed by the human body. After extracting collagen by enzymatic hydrolysis, the molecular chain is cut into polypeptides by soaking and stirring in glacial acetic acid, which can control the molecular weight of collagen below 3000 daltons. Collagen and its hydrolysate have a similar collagen structure to artificial skin. Due to the presence of a large number of hydrophilic groups in the collagen molecule, it has good moisturizing effect and can achieve the purpose of keeping the skin moist.

[0005] A paper (Reference: Extraction and evaluation of moisture absorption and retention properties of fish swim bladder glycosaminoglycans. Food Industry Science and Technology, 2017, 38(16): 118-125) reported the optimal process conditions for enzymatic extraction of fish swim bladder glycosaminoglycans: using largemouth bass swim bladder as raw material, solid-liquid ratio 1:20 g:mL, enzyme hydrolysis time 4 h, enzyme hydrolysis temperature 50°C, enzyme dosage 7%, and enzyme hydrolysis pH 8. Under the optimal conditions, the yield of fish swim bladder glycosaminoglycan crude product was 1.19%, and the glycosaminoglycan content was 19.09%. Fish swim bladder glycosaminoglycans have good moisture absorption and retention properties, and the overall moisture absorption and retention properties are better than those of chitosan and sodium alginate, which are common moisturizers.

[0006] In summary, fish swim bladder extract has certain research basis and feasibility as a hydrophilic cosmetic raw material. However, the preparation of fish swim bladder extract in existing public literature has certain limitations, i.e., only the preparation of a single type of substance (i.e., collagen or glycosaminoglycan) and its properties as a cosmetic raw material are concerned, and no research results on the preparation method and properties of fish swim bladder containing multiple functional substances (i.e., collagen, glycosaminoglycan, and glycosaminoglycan) are reported.

[0007] The transdermal absorption in cosmetics refers to the process that the functional ingredients in cosmetics act on the skin surface or enter the epidermis or dermis, and accumulate and play a role at the site according to the effectiveness of the product. Studies have shown that the addition of a large number of nutritional ingredients in skin care products, no matter what essence or nutrition, if the skin cannot absorb it, it is a burden. The excess of cosmetic nutritional ingredients on the skin surface is one of the important reasons for the "skin oxidation", which can cause premature aging of the skin, degradation of metabolic function, dry, sensitive, wrinkled, pigmented, acne and the like. Moreover, a large amount of vitamins, proteins and biological cell nutrients are needed for the growth and reproduction of bacteria on the skin surface, which are the main components of nutritional cosmetics. If the nutritional ingredients of the cosmetic cannot be completely absorbed by the skin, it will become a breeding ground for parasitic bacteria, and a large number of bacteria will also cause skin infection. Therefore, the importance of transdermal absorption in cosmetics (Reference: Efficacy, Mechanism of Action and Application Research of Chinese Medicine Transdermal Absorption Promoters in Cosmetics. Beijing University of Business and Technology, 2010. Master's Thesis). Therefore, it is of important application significance to improve the transdermal absorption of cosmetic raw materials. SUMMARY

[0008] The present application aims to provide a kind of biological material with hydrophilicity and promoting transdermal absorption, uses fish swim bladder extract rich in collagen, elastin and glycosaminoglycan as hydrophilic substance, by with the transdermal absorption of specific amino acid sequence of promoting polypeptide complex, thus obtain the dual function of hydrophilic and promoting transdermal absorption, make it better application in biological material manufacturing field, especially in the field of cosmetics, for this purpose, the present application also provides the method for preparing the biological material and the cosmetic containing the biological material.

[0009] The present application is realized by the following technical scheme: a kind of biological material with hydrophilicity and promoting transdermal absorption, comprising the following mass percentage concentration components: fish swim bladder extract 95% and transdermal absorption promoting polypeptide 5%,

[0010] The fish swim bladder extract is a hydrophilic substance containing collagen, elastin and glycosaminoglycan extracted from fish swim bladder; the amino acid sequence of the transdermal absorption promoting polypeptide is GGPAAVSSQ or AAVPPLTTC.

[0011] Specifically, the transdermal absorption promoting polypeptide with amino acid sequence GGPAAVSSQ corresponds to the three-letter amino acid sequence Gly-Gly-Pro-Ala-Ala-Val-Ser-Ser-Gln, and the structural formula satisfies formula (1):

[0012]

[0013] The transdermal absorption promoting polypeptide with amino acid sequence of AAVPPLTTC corresponds to the three-letter amino acid sequence of Ala-Ala-Val-Pro-Pro-Leu-Thr-Thr-Cys, and the structural formula satisfies the following formula (2):

[0014]

[0015] The fish mucus extract contains collagen 45-55%, elastin 7-11%, glycosaminoglycan 8-10% and water 10-30% by mass percentage.

[0016] The fish mucus is selected from any one of grass carp, carp, crucian carp, silver carp, eel or sturgeon.

[0017] A preparation method of a biological material with both hydrophilicity and transdermal absorption promoting property, 95% of fish mucus extract and 5% of transdermal absorption promoting polypeptide are compounded, and then are crushed and stirred uniformly to obtain a powder-like biological material,

[0018] The fish mucus extract is a hydrophilic material containing collagen, elastin and glycosaminoglycan extracted from fish mucus; and the amino acid sequence of the transdermal absorption promoting polypeptide is GGPAAVSSQ or AAVPPLTTC.

[0019] During extraction, the cleaned fish mucus is crushed and then water is removed, and then sodium hydroxide is used for decomposition, and after decomposition, centrifugation, screening, pH adjustment, drying and crushing, the fish mucus extract is obtained.

[0020] During sodium hydroxide decomposition, the weight ratio of fish mucus material to sodium hydroxide is controlled to be 1:3.

[0021] During sodium hydroxide decomposition, the mixture of fish mucus material and sodium hydroxide is sealed, and then water bath heating is adopted, the heating temperature is controlled to be 40-50 DEG C, and the decomposition time is controlled to be 1-24 hours.

[0022] A cosmetic with both hydrophilicity and transdermal absorption promoting property contains the above biological material.

[0023] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0024] (1) The present application uses the compounding of fish mucus extract and customized transdermal absorption promoting polypeptide, so that the cosmetic raw material has both hydrophilicity and transdermal absorption promoting property, which is more conducive to its application in cosmetics, improves the absorption of hydrophilic substances by the skin, and provides good moisturizing effect.

[0025] (2) The existing fish swim bladder extract preparation technology (paper: Research on extraction process and properties of collagen from common carp skin and swim bladder. Wuhan Polytechnic University, 2012. Master's thesis; Extraction of swim bladder glycosaminoglycans and evaluation of its moisture absorption and retention properties. Food Industry Science and Technology, 2017, 38(16): 118-125; Patent: Sturgeon swim bladder protein peptide and its application in antioxidant active substances, application number 202310169787.3), mainly adopts biological enzyme decomposition and purification process, and only focuses on the preparation of a single type of material (i.e. collagen or glycosaminoglycan) and its properties as a cosmetic raw material. Therefore, the method of the present application uses sodium hydroxide decomposition method for fish swim bladder extract, and the prepared material contains high percentage of collagen, elastin and glycosaminoglycan (collagen 45-55%, elastin 7-11%, glycosaminoglycan 8-10%) at the same time. The synergistic effect of the three functional components can improve the hydrophilicity of the material, and has the characteristics of low extraction cost and less pollution, which is beneficial to large-scale industrial production.

[0026] (3) The present application provides a customized transdermal absorption promoting polypeptide to promote the transdermal absorption performance of fish swim bladder extract. The polypeptide structure contains both hydrophobic and hydrophilic segments, which can give the cosmetic raw material more obvious transdermal absorption promoting property, without the need for additional transdermal absorption promoters, and is safe to use. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The preparation process diagram of the hydrophilic cosmetic raw material (i.e. fish swim bladder extract).

[0028] Figure 2 The test result diagram of the material hydrophilicity (water drop penetration time).

[0029] Figure 3 The test process diagram of the material hydrophilicity (moisture absorption and retention).

[0030] Figure 4 The test result diagram of the material hydrophilicity (moisture retention).

[0031] Figure 5 The test result diagram of the material hydrophilicity (moisture absorption).

[0032] Figure 6 The transdermal penetration test process diagram. DETAILED DESCRIPTION

[0033] The invention purpose, technical scheme and beneficial effects of the present application will be further described in detail as follows.

[0034] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the invention as claimed, unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0035] The present application aims to provide a biomaterial prepared by compounding fish swim bladder extract and a transdermal absorption promoting polypeptide, which can be used as a raw material for cosmetics. The fish swim bladder extract is prepared by sodium hydroxide decomposition, which can enrich the hydrophilic active ingredients in the fish swim bladder extract, and obtain high percentage concentration content of collagen, elastin and glycosaminoglycan. The transdermal absorption promoting polypeptide is a customized polypeptide for the fish swim bladder extract, which has the structural characteristics of hydrophobic segment and hydrophilic segment, and can give the fish swim bladder extract better transdermal absorption promotion. Therefore, the biomaterial of the present application has both hydrophilicity and transdermal absorption promotion, and considering the specific preparation process of the fish swim bladder extract and its hydrophilic function in the field of cosmetics, the present application also provides a preparation method of the biomaterial and a cosmetic containing the biomaterial.

[0036] Further, the technical solution of the present application can be summarized as follows:

[0037] Fish swim bladder extract: a hydrophilic material rich in collagen, elastin and glycosaminoglycan extracted from fish swim bladder. In preparation, fish swim bladder can use any one of grass carp, carp, crucian carp, silver carp, eel or sturgeon. Taking grass carp swim bladder as an example, the extraction process of fish swim bladder extract can include the following steps:

[0038] 1) Take fresh grass carp swim bladder and thaw in purified water, soak for about 2 hours, then wash the residual blood and other residues on the grass carp swim bladder with purified water.

[0039] 2) Grind the thawed and cleaned fish swim bladder with a grinder for about 10 seconds.

[0040] 3) Dry the ground fish swim bladder material on a screen for 30 minutes, then place it in a drying oven at 20-50℃ for 30-60 minutes to remove most of the water on its surface.

[0041] 4) Weigh the fish swim bladder material, then decompose it with 0.5-3M sodium hydroxide, and mix according to the ratio of biological tissue to sodium hydroxide of 1:3 (weight ratio).

[0042] 5) Place the mixed solution in a beaker and seal it, then place it in a water bath at 40-50℃ for 1-24 hours. During decomposition, the water bath liquid surface should be higher than or flush with the sample liquid surface.

[0043] 6) Take out the mixed solution of the decomposed swim bladder and divide it into centrifuge tubes for centrifugation at 2000 rpm for 10 minutes.

[0044] 7) After centrifugation, take out the supernatant and pass it through a 60-mesh stainless steel screen.

[0045] 8) Adjust the pH of the screened sample solution to 6.5-7.4 using 5% hydrochloric acid.

[0046] 9) Put the sample solution into a container, and then perform air blowing drying to remove water, with a drying temperature of 40-50°C and a drying time of 12-24 hours.

[0047] 10) After drying, take out the sample, crush it using a crusher, pass it through a 60-mesh stainless steel screen, and obtain a powder-like swim bladder extract, which is sealed in a bag and stored in a refrigerator.

[0048] The swim bladder extract thus prepared contains collagen 45-55%, elastin 7-11%, glycosaminoglycan 8-10%, and water 10-30% (by mass percentage concentration).

[0049] The polypeptide for promoting transdermal absorption includes two kinds, one of which has an amino acid sequence of GGPAAVSSQ, corresponding to a three-letter amino acid sequence of Gly-Gly-Pro-Ala-Ala-Val-Ser-Ser-Gln, and a structural formula as shown in the following formula (1):

[0050]

[0051] The other polypeptide for promoting transdermal absorption has an amino acid sequence of AAVPPLTTC, corresponding to a three-letter amino acid sequence of Ala-Ala-Val-Pro-Pro-Leu-Thr-Thr-Cys, and a structural formula as shown in the following formula (2):

[0052]

[0053] As can be seen from the above structure, the polypeptide for promoting transdermal absorption of the present application simultaneously contains a hydrophobic segment and a hydrophilic segment. This custom polypeptide with the structural characteristics of simultaneously containing a hydrophobic segment and a hydrophilic segment endows the cosmetic raw material with the property of promoting transdermal absorption. The polypeptide for promoting transdermal absorption with an amino acid sequence of GGPAAVSSQ has a hydrophobic segment of GGPAAV and a hydrophilic segment of SSQ; and the polypeptide for promoting transdermal absorption with an amino acid sequence of AAVPPLTTC has a hydrophobic segment of AAVPPL and a hydrophilic segment of TTC.

[0054] The fish mucus extract and the transdermal absorption promoting polypeptide are weighed according to the mass percentage concentration, and the fish mucus extract is 95% and the transdermal absorption promoting polypeptide is 5%. Then, the fish mucus extract and the transdermal absorption promoting polypeptide are crushed by a crusher and stirred to obtain a powder sample, which is the biomaterial of the application. The biomaterial can be added as an effective substance in cosmetics, or mixed with other conventional auxiliaries to prepare cosmetics, so that the cosmetics have both hydrophilicity and transdermal absorption promoting property.

[0055] The application will be further described in detail below in combination with examples, but the embodiments of the application are not limited thereto.

[0056] Example 1

[0057] This example relates to a biomaterial, in particular a raw material for cosmetics, which comprises the following components and amounts: fish mucus extract 95% and transdermal absorption promoting polypeptide 5%.

[0058] In this example, the grass carp mucus is used as a raw material, and the fish mucus extract is obtained according to the above preparation steps. The specific drying temperature in step 3) is 50°C, and the time is 30 minutes. In step 4), 3M sodium hydroxide is used for decomposition. In step 5), the decomposition is performed in a water bath at 40°C for 1 hour. In step 8), the pH of the adjusted sample solution is 6.5. In step 9), the drying temperature is 40°C, and the time is 12 hours. For details, see Figure 1 the preparation process diagram of the fish mucus extract shown in Figure 1 In the above-mentioned fish mucus extract preparation process diagram, A is the initial state of the fish mucus raw material, B is the state after preliminary crushing and washing by a crusher, C is the initial state of decomposition by sodium hydroxide solution, D is the state after 0.5 hours of alkali decomposition, E is the state after 1 hour of alkali decomposition, F is the state after centrifugation, and G is the state after drying.

[0059] The fish mucus extract and the transdermal absorption promoting polypeptide (amino acid sequence GGPAAVSSQ, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) are weighed according to the ratio and then compounded together. Then, the fish mucus extract and the transdermal absorption promoting polypeptide are crushed by a crusher and stirred to obtain a powder sample, which is the biomaterial.

[0060] Example 2

[0061] This example relates to a biomaterial, in particular a raw material for cosmetics, which comprises the following components and amounts: fish mucus extract 95% and transdermal absorption promoting polypeptide 5%.

[0062] In this example, the preparation steps of the fish mucus extract are the same as those in Example 1, and the only difference is that 1M sodium hydroxide is used for decomposition in step 4), and the decomposition time in step 5) is 12 hours.

[0063] The fish swim bladder extract and the transdermal absorption promoting polypeptide (amino acid sequence AAVPPLTTC, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) are weighed according to the proportion, then compounded together, then crushed by a crusher and stirred to obtain a powder-like biomaterial.

[0064] Test Example 1: Detection of Collagen Content in Fish Swim Bladder Extract

[0065] Experimental detection method: Refer to the collagen content detection method described in the paper (Glycidyl methacrylate-crosslinked fish swimbladder as a novel cardiovascular biomaterial with improved antithrombotic and anticalcification properties. Journal of biomaterials applications, 2022, 36(7), 1188-1200).

[0066] The test sample is the fish swim bladder extract prepared in Example 1 and Example 2.

[0067] Hydroxyproline was determined using a hydroxyproline kit (Nanjing Jiancheng Biological Engineering Institute). The kit utilizes the property that the oxidation product of hydroxyproline can react with dimethylaminobenzaldehyde to produce a purple red color, and quantitatively analyzes the hydroxyproline in the material, which can indirectly measure the content of collagen.

[0068] The specific experimental steps are as follows:

[0069] 1. Hydrolyze the sample: weigh about 50 mg of sample, use the hydrolysis solution in the kit to hydrolyze the tissue in a centrifuge tube, about 1 ml is needed. Tighten the lid of the centrifuge tube containing the hydrolysis solution, fix it with a foam holder, and put it into a water bath at 95°C for 20 minutes;

[0070] 2. After the water bath, the solution in the centrifuge tube is clear, take out the centrifuge tube and use cold water to cool the tube wall to room temperature. In order to adjust the pH value later, add 10 μL of indicator and mix well;

[0071] 3. Adjust pH: first add 1 mL of solution A, cover the lid and mix well, the solution will turn red. Then slowly add solution B until the solution turns yellow-green. Note that each drop should be mixed well and observed for color change to prevent over-dosing;

[0072] 4. Use double distilled water to make the solution to 10 mL;

[0073] 5. Take 3 mL from the 10 mL solution to a new centrifuge tube, add about 20 mg of activated carbon, and centrifuge at 3000 rpm for 10 minutes. The supernatant should be colorless and transparent at this time. Take 1 mL of the supernatant for subsequent quantitative testing.

[0074] 6. Prepare a blank control: add 1 mL of double distilled water to the blank tube;

[0075] 7. Prepare a standard control: add 1 mL of 5 μg / mL standard application liquid to the standard tube;

[0076] 8. Prepare a sample to be tested: add 1 mL of the sample to be tested obtained in the previous five steps to the sample tube;

[0077] 9. Add 0.5 mL of reagent one to the blank tube, standard tube, and sample tube, cover the cap, shake to mix well, and stand for 10 minutes;

[0078] 10. Add 0.5 mL of reagent two to the blank tube, standard tube, and sample tube, cover the cap, shake to mix well, and stand for 5 minutes;

[0079] 11. Add 0.5 mL of reagent three to the blank tube, standard tube, and sample tube, mix well, cover the cap, and place in a water bath at 60°C for 15 minutes;

[0080] 12. After the water bath, rinse the tube wall with cold water to cool it, and centrifuge at 3500 rpm for 10 minutes;

[0081] 13. Take 200 μL of the supernatant to a microplate, and measure the absorbance value at a wavelength of 550 nm;

[0082] 14. Calculate the hydroxyproline content using the absorbance values of the blank, standard, and determination, the content of the standard, the total volume of the hydrolysis liquid, and the weight of the material.

[0083] Test results: The collagen content in the swim bladder extract prepared in Example 1 and Example 2 was in the range of 45-55%.

[0084] Test Example 2: Detection of elastin content in swim bladder extract

[0085] Experimental detection method: The elastin content detection method described in the paper (Glycidyl methacrylate-crosslinked fish swimbladder as a novel cardiovascular biomaterial with improved antithrombotic and anticalcification properties. Journal of biomaterials applications, 2022, 36(7), 1188-1200) was used.

[0086] The test sample was the fish swim bladder extract prepared in Example 1 and Example 2.

[0087] The determination of elastin content used the Fastin kit (Biocolor Life Science, UK) to determine the elastin content of the pericardium according to the product instructions. The Fastin elastin assay is a quantitative dye-binding method using the dye 5, 10, 15, 20-tetraphenyl-21H, 23H-porphine tetrasulfonate (TPPS). The elastin in the sample was first extracted. The weighed sample was placed in a 1.5 mL microcentrifuge tube and 750 μL of 0.25 M oxalic acid was added. The tube was placed in a metal heating block with the thermostat set to 100°C for 60 minutes. After cooling to room temperature, it was centrifuged at 10,000 rpm for 10 minutes. The liquid was aspirated with a pipette and the extract was retained in a labeled container for analysis. Another 750 μL of 0.25 M oxalic acid was added to the residual tissue in the test tube and heated again for 60 minutes. The volume of the extract was recorded in order to calculate the elastin content of the tissue. A set of 1.5 mL microcentrifuge tubes was labeled in the following three ways and the corresponding reagents were added:

[0088] 1. Reagent blank: 100 μL of test solution solvent (buffer / PBS / water / 0.25 M oxalic acid);

[0089] 2. Alpha-elastin standard: Volumes of 12.5, 25.0 and 50.0 μL;

[0090] 3. Test sample: 50 μL to 250 μL of tissue extract from in vivo sources.

[0091] Add an equal volume of elastin precipitation reagent to each tube. Cap the tubes and vortex briefly to mix the contents, and let stand for 15 minutes to allow complete precipitation of the alpha-elastin. Centrifuge the tubes at 10,000g for 10 minutes. Gently remove the liquid from the tubes. The next step is to form the elastin-dye complex. Add 1.00 mL of dye reagent to all tubes. Cap the tubes and invert the tubes to mix the contents. Then disperse the elastin precipitate using a vortex mixer. Place the tube rack on a mechanical shaker and allow the reaction between the elastin and the dye to proceed for 90 minutes. Centrifuge the tubes at 10,000g for 10 minutes. Then recover the elastase-dye complex. Drain the unbound dye tubes. The elastin-dye complex can be observed as a reddish-brown deposit at the bottom and inner lower wall of the tubes. After that, release and recover the elastin-bound dye. Add 250 μL of dye dissociation reagent to each tube. Cap the tubes and use a vortex mixer to release the dye into solution. Repeat the vortex mixing after 10 minutes to ensure that all bound dye has entered solution. Transfer the contents of each tube to a well on a 96-well flat-bottom microplate. Finally, determine the elastin content. Place the microplate in a microplate reader. Select a wavelength of 513 nm. Plot the reference standards, and use this plot to determine the elastin content of the test samples.

[0092] Test results: The detection results of the elastin content in the swim bladder extracts prepared in Example 1 and Example 2 range from 7 to 11%.

[0093] Test Example 3: Detection of glycosaminoglycan content in swim bladder extract

[0094] Experimental detection method: Refer to the glycosaminoglycan content detection method described in the paper (Glycidyl methacrylate-crosslinked fish swimbladder as a novel cardiovascular biomaterial with improved antithrombotic and anticalcification properties. Journal of biomaterials applications, 2022, 36(7), 1188-1200).

[0095] Test samples are the swim bladder extracts prepared in Example 1 and Example 2.

[0096] Glycosaminoglycan quantification was performed using the Blyscan kit (Biocolor Life Science, UK). The dye tag used in this assay was 1,9-dimethylmethylene blue. A papain extraction reagent was first prepared. To 50 mL of 0.2 M sodium phosphate buffer at pH 6.4, 400 mg of sodium acetate, 200 mg of EDTA disodium salt, and 40 mg of cysteine hydrochloride were added. After the above ingredients were dissolved in the buffer, 250 ul of a papain enzyme suspension was added, which contained approximately 5 mg of enzyme. The glycosaminoglycans were extracted from the tissue sample using the papain extraction reagent. The test sample (20-50 mg) and 1 mL of the papain extraction reagent were placed in a 1.5 mL labeled centrifuge tube. The centrifuge tube was placed in a temperature-regulated metal heating block or water bath at 65 °C. The digestion time was approximately 3 hours. The tube was centrifuged at 10,000 g for 10 minutes. The supernatant was decanted for subsequent testing. The test sample was then prepared for testing. A series of 1.5 mL centrifuge tubes were labeled, all of the test sample tubes, standard reference tubes, and blank control tubes were prepared as follows:

[0097] 1. Blank control tube: 100 μL of deionized water or test sample buffer;

[0098] 2. Standard reference tubes: 1 μg, 2 μg, 3 μg, 4 μg, and 5 μg of the standard reference sample were each weighed and quantitatively transferred to 100 μL using the same solvent as the blank control tube;

[0099] 3. Test sample tube: approximately 50 μL of the sample was added, and deionized water or an appropriate buffer was used to adjust the total volume of the test sample tube to 100 μL.

[0100] The test sample was then processed. Blyscan dye reagent was added to each centrifuge tube at 1 mL. The centrifuge tubes were capped, inverted repeatedly to mix the contents, and then placed on a mechanical shaker for 30 minutes. During this time, the glucosamine-dye complex was formed, and the dye was converted from a soluble state to a precipitate. The tubes were centrifuged at 12,000 rpm for 10 minutes. The tubes were inverted onto absorbent paper, and the tubes were tapped to remove the supernatant (which contained unbound dye) and to keep the precipitate tightly attached to the bottom of the tube. The dye was then dissociated from the glycosaminoglycans. To each tube, dissociation reagent was added at 0.5 mL, the tubes were recapped, and the samples were mixed using a vortex mixer. After approximately 10 minutes, the dye was completely dissolved. The tubes were centrifuged at 12,000 rpm for 5 minutes to remove the foam, and the tubes were kept capped until the absorbance was measured. Finally, the test sample was tested. 200 μL of the sample was transferred to a 96-well microplate, the spectrophotometer was adjusted to a wavelength of 656 nm, and the absorbance of the blank control, standard reference, and test sample was measured. The glycosaminoglycan concentration of the sample was read from the standard curve.

[0101] Test results: The glycosaminoglycan content of the swim bladder extracts prepared in Example 1 and Example 2 was in the range of 8% to 10%.

[0102] Test Example 4: Test for material hydrophilicity (water drop penetration time)

[0103] Experimental detection method: Reference literature as follows: Excimer laser induced PET material surface grafting to improve surface hydrophilicity, Zhu Min, Donghua University, doctoral dissertation, 2006.

[0104] Test samples include: grass carp swim bladder extract in Example 1, control samples include pig blood vessel extract (prepared by the same method as Example 1, only the raw material is replaced by pig blood vessel), collagen standard.

[0105] Sample preparation: First crush with a crusher, and then use a manual tablet press to make a round piece with a diameter of 1 centimeter (height of about 0.1 centimeters or less, determined according to the total amount of the sample).

[0106] Experimental procedure: Stick a 10 uL drop of deionized water to the surface of the material, and start timing until the water drop penetrates and the material structure is fully exposed. This time is the water drop penetration time, measured in seconds. The above tests were completed in a constant temperature and humidity chamber with constant temperature and humidity conditions of 20±1℃ and relative humidity of 65±2%.

[0107] See the material hydrophilicity (water drop penetration time) test results shown in Figure 2 Figure 2 , where A is the collagen standard, B is the pig blood vessel extract, and C is the swim bladder extract.

[0108] Experimental results: The water drop penetration time of collagen is 370s, the water drop penetration time of pig blood vessel extract is 280s, and the water drop penetration time of swim bladder extract is 264s. That is, the results show that the water drop penetration time of collagen is longer, that is, the hydrophilicity of swim bladder extract is better than that of collagen.

[0109] Test Example 5: Test for material hydrophilicity (moisture absorption and retention)

[0110] Experimental detection method: Reference literature as follows: Research on the moisturizing and skin care effect of cod skin collagen peptide, Li Xing, Ocean University of China, master's degree thesis, 2014.

[0111] Test samples include: grass carp swim bladder extract in Example 1, control samples include pig blood vessel extract (prepared by the same method as Example 1, only the raw material is replaced by pig blood vessel), collagen standard.

[0112] ​Sample preparation: First, crush with a pulverizer, pass through a 60-mesh stainless steel sieve, and use a hand-operated tablet machine to make a 1-centimeter-diameter disc (height of about 0.1 centimeters or less, determined according to the total amount of the sample).

[0113] Experimental procedure:

[0114] Determination of hygroscopicity:

[0115] Place the material in an oven at 50°C and dry thoroughly (overnight). At a constant temperature of 25°C, maintain a relative humidity (RH) of 81% in a foam box with a saturated ammonium sulfate aqueous solution, accurately weigh 0.2 g of the sample, and place it in the foam box with a relative humidity (RH) of 81%, using glycerol as a control. Accurately weigh each sample at regular intervals until it is removed after 31 hours.

[0116] Hygroscopicity (%) = (Wn - Wo) / Wo x 100%

[0117] Wo is the mass of the sample before hygroscopicity, and Wn is the mass of the sample after hygroscopicity.

[0118] Determination of moisture retention:

[0119] Accurately weigh 0.2 g of the dried sample, add 10% by mass of water, and then place it in a foam box with a relative humidity (RH) of 81%, using glycerol as a control. Accurately weigh each sample at regular intervals until it is removed after 31 hours.

[0120] Moisture retention rate (%) = Hn / Ho x 100%

[0121] Ho is the water content of the sample before placement, and Hn is the water content of the sample after a certain period of time.

[0122] For details, see Figure 3 the material hydrophilicity (hygroscopicity and moisture retention) detection process diagram, Figure 3 where A is the initial state of hygroscopicity and moisture retention, B is the state after 6 hours of hygroscopicity and moisture retention, C is the state after 23 hours of hygroscopicity and moisture retention, and D is the state after 31 hours of hygroscopicity and moisture retention.

[0123] Experimental results: see Figure 4 , Figure 5 and Table 1 below.

[0124] Table 1: Original data for hygroscopicity and moisture retention.

[0125]

[0126]

[0127] As can be seen, the moisture retention and hygroscopicity of the swim bladder extract of Example 1 are both better than those of the collagen standard.

[0128] Test Example 6: Test for Transdermal Penetration of Materials

[0129] Experimental detection method: Reference literature as follows: Zhu Min. Research and development of anti-aging cosmetics based on tea oil [D]. Hefei University of Technology, 2017.

[0130] Test samples include: powder samples obtained by compounding 95% of the swim bladder extract in Example 1 and 5% of the transdermal absorption promoting polypeptide (amino acid sequence GGPAAVSSQ, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.).

[0131] Experimental steps:

[0132] Specific steps are as follows:

[0133] 1. Take fresh pigskin, scrape off the oil on the pigskin, and trim the pigskin into a round piece with a diameter of 2 cm and a thickness of 1 mm using surgical scissors and a small knife;

[0134] 2. Take 0.5 g of sample, add 2 mL of pure water, adjust the pH to neutral with 5% hydrochloric acid, and dilute to 5 mL with pure water;

[0135] 3. Take the diffusion cell, add 5.3 mL of pure water to the lower layer, place the pigskin in the middle of the two layers of the diffusion cell, and fix it tightly with a clamp, and add 0.6 mL of 10% sample solution to the upper layer, and seal it with plastic wrap;

[0136] 4. Place the sample-containing diffusion cell in a water bath and react at 37°C for 24 hours. After the reaction is completed, collect the solutions in the upper and lower layers.

[0137] The contents of collagen, elastin and glycosaminoglycans in the lower layer solution were analyzed (refer to the test methods described in Test Examples 1-3 above). The penetration promoting ratio was calculated according to the following formula:

[0138] Penetration promoting ratio = penetration amount (with penetration promoter) / penetration amount (without penetration promoter)

[0139] For details, see Figure 6 the transdermal penetration test process diagram, Figure 6 wherein A is a diffusion cell device diagram, B is a test sample placement diagram before water bath, C is a penetration diagram in water bath, D is a state after water bath, and E is a lower layer solution collection diagram.

[0140] Experimental results: Compared with the control sample (containing the swim bladder extract in Example 1), the penetration promoting ratio of the three functional ingredients in the powder sample of Example 1 (obtained by compounding 95% of the swim bladder extract and 5% of the transdermal absorption promoting polypeptide with an amino acid sequence of GGPAAVSSQ) is shown in the following Table 2:

[0141] Table 2: The penetration promotion ratio of three functional components

[0142] Functional classification by class Promote permeability ratio Collagen 4.71 Elastin 3.26 Glycosaminoglycan 6.65

[0143] From the above table 2, it can be seen that the complexing of the fish swim-bladder extract in example 1 to promote the transdermal absorption of polypeptides can promote the transdermal penetration of the three functional components in the fish swim-bladder extract, i.e. collagen, elastin and glycosaminoglycan.

[0144] Of course, the biomaterials described in the present application can also be used as dressing products in medical devices due to their hydrophilicity and promotion of transdermal absorption, and have a wide market prospect.

[0145] The above description is only a preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification or equivalent change made according to the technical essence of the present application to the above embodiment falls within the protection scope of the present application.

Claims

1. A biomaterial having both hydrophilicity and promoting transdermal absorption, characterized in that: Contains the following components by mass percentage: 95% fish maw extract and 5% peptide that promotes transdermal absorption. Fish maw extract is a hydrophilic substance containing collagen, elastin and glycosaminoglycans obtained by extracting fish maw as raw material; the amino acid sequence of the polypeptide that promotes transdermal absorption is GGPAAVSSQ or AAVPPLTTC.

2. The biomaterial according to claim 1, characterized in that: The structural formula of the polypeptide promoting transdermal absorption with the amino acid sequence of GGPAAVSSQ satisfies the following formula (1):

3. The biomaterial according to claim 1, wherein: The structural formula of the polypeptide promoting transdermal absorption with the amino acid sequence of AAVPPLTTC satisfies the following formula (2):

4. The biomaterial according to claim 1, wherein: Calculated by mass percentage, the fish maw extract contains 45-55% collagen, 7-11% elastin, 8-10% glycosaminoglycan and 10-30% water.

5. The biomaterial according to claim 1, wherein: The fish maw is selected from any one of grass carp, carp, crucian carp, silver carp, eel or sturgeon.

6. A method for preparing a biomaterial having both hydrophilicity and enhanced transdermal absorption, characterized in that: According to the concentration percentage by mass, 95% of fish maw extract and 5% of polypeptide for promoting transdermal absorption are compounded, crushed and stirred evenly to obtain a powdered biological material. Fish maw extract is a hydrophilic substance containing collagen, elastin and glycosaminoglycans obtained by extracting fish maw as raw material; the amino acid sequence of the polypeptide that promotes transdermal absorption is GGPAAVSSQ or AAVPPLTTC.

7. The preparation method according to claim 6, characterized in that: During the extraction, the cleaned fish maw is crushed to remove moisture, and then decomposed with sodium hydroxide. After decomposition, the fish maw extract is obtained by centrifugation, sieving, adjusting pH, drying, and crushing.

8. The preparation method according to claim 7, characterized in that: When sodium hydroxide is decomposed, the weight ratio of the fish maw material to the sodium hydroxide is controlled to be 1:

3.

9. The preparation method according to claim 7, characterized in that: When sodium hydroxide is used for decomposition, the mixed solution of the fish maw material and sodium hydroxide is sealed and then heated in a water bath. The heating temperature is controlled at 40-50° C., and the decomposition time is controlled at 1-24 hours.

10. A cosmetic having both hydrophilicity and the ability to promote transdermal absorption, characterized in that: Containing the biomaterial according to any one of claims 1 to 5.

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