Paa boulengeri skin antibacterial peptide extract, paa boulengeri skin antibacterial repair gel and preparation method of paa boulengeri skin antibacterial peptide extract

Abstract

The invention discloses a paa boulengeri skin antibacterial peptide extract, paa boulengeri skin antibacterial repair gel and a preparation method of the paa boulengeri skin antibacterial peptide extract, and belongs to the technical field of biology. The preparation method of the paa boulengeri skin antibacterial peptide extract comprises the following steps: performing ultrasonic extraction on dried paa boulengeri skin at 60-70 DEG C by taking an acetic acid solution as an extracting solution, performing post-treatment after the extraction is completed, and performing freeze drying after the post-treatment is completed to obtain the paa boulengeri skin antibacterial peptide extract. The antibacterial peptide is extracted through the ultrasonic-assisted acidolysis method of the paa boulengeri skin, so that the use of a solvent is reduced, the degradation of the antibacterial peptide in the extraction process is reduced, the extraction rate is increased, and the stability of the peptide is favorably maintained. The antibacterial peptide is freeze-dried, and moisture is removed, so that the stability of the antibacterial peptide is maintained.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an antibacterial peptide extract from Rana spinosa skin, an antibacterial repair gel from Rana spinosa skin and a preparation method thereof. Background Art

[0002] Spinosaurus spp. Quasipaa boulengeri ) is an amphibian of the family Forked Glossidae and genus Spinothoracidae. The muscle of the spiny-bellied frog is rich in 16 essential amino acids, with higher levels than those of the Chinese soft-shelled turtle and the snakehead fish. The muscle of the spiny-bellied frog is more tender and palatable than that of the bullfrog. Besides being edible, the spiny-bellied frog also has medicinal and other uses. It can clear the heart and moisten the lungs, effectively alleviating symptoms such as lung discomfort and irritability caused by dryness and heat. Its yin-nourishing and fire-reducing properties make it a natural remedy for regulating the body's yin and yang balance, making it particularly suitable for those with yin deficiency and hyperactivity of fire. Its spleen-tonifying and stomach-strengthening properties help promote digestion and absorption, strengthening the spleen and stomach function. Its tonifying effects can help the weak recover their vitality. Its heat-removing and toxin-removing properties can alleviate various discomforts caused by the accumulation of heat and toxins.

[0003] Research has shown that antimicrobial peptides extracted from the skin of Rana spinosa possess broad-spectrum antimicrobial activity, inhibiting the growth of a wide range of bacteria. When antimicrobial peptides come into contact with bacteria, they first bind to negatively charged sites on the bacterial cell membrane through electrostatic interactions. Due to their unique structure, antimicrobial peptides can form pores in the cell membrane, disrupting its integrity and causing the outflow of intracellular substances, ultimately rendering the bacteria unable to maintain normal physiological functions and causing their death. Furthermore, antimicrobial peptides may penetrate into bacterial cells and interfere with key physiological processes such as DNA replication and protein synthesis, thereby disrupting the bacteria's survival from within.

[0004] However, the antimicrobial peptide from the skin of R. spinosa can be degraded by environmental factors (such as oxidation and high temperature), which can weaken its antimicrobial effect. Therefore, it is necessary to provide a method for preparing the antimicrobial peptide that can improve its stability. Summary of the Invention

[0005] To address the above-mentioned issues, the present invention provides an antimicrobial peptide extract from Rana spinosa skin, an antimicrobial repair gel from Rana spinosa skin, and methods for preparing the same. This method utilizes ultrasound-assisted acid hydrolysis of Rana spinosa skin to extract the antimicrobial peptides, reducing solvent usage and degradation during the extraction process, thereby increasing extraction yield and maintaining peptide stability. The antimicrobial peptides are freeze-dried to remove moisture, thereby maintaining their stability.

[0006] To achieve the above object, the technical solution adopted by the present invention is: The first object of the present invention is to provide a method for preparing an antimicrobial peptide extract from the skin of Rana spinosa, comprising the following steps: The dried skin of Rana spinosa was ultrasonically extracted at 60℃~70℃ using acetic acid solution as the extraction solution, and post-processing was performed after the extraction was completed. After the post-processing was completed, freeze-drying was performed to obtain the antimicrobial peptide extract from the skin of Rana spinosa.

[0007] In a preferred embodiment of the present invention, the ratio of dried Rana spinulosa skin to acetic acid solution is 1 g: 50 ml to 100 ml, and the concentration of the acetic acid solution is 0.105 g / ml to 0.21 g / ml.

[0008] In a preferred embodiment of the present invention, the extraction time is 60 min to 120 min.

[0009] In a preferred embodiment of the present invention, the ultrasonic power is 500W~600W.

[0010] The second object of the present invention is to provide the antimicrobial peptide extract from the skin of Rana spinosa prepared by the above preparation method.

[0011] The third object of the present invention is to provide an antibacterial repair gel of Rana spinosa skin, which is prepared by extracting antibacterial peptides from Rana spinosa skin.

[0012] A fourth object of the present invention is to provide a method for preparing the aforementioned Rana spinosa skin antibacterial repair gel, comprising the following steps: Glycerol and sodium carboxymethyl β-glucan are mixed evenly to obtain a first mixed solution; trehalose, disodium edetate and water are mixed evenly to obtain a second mixed solution; butanediol, 1,2-hexanediol, phenoxyethanol and PEG-40 hydrogenated castor oil are mixed evenly to obtain a third mixed solution.

[0013] Under the condition of 80°C~90°C, the second mixed solution and the first mixed solution of sodium carboxymethyl β-glucan and disodium edetate are added to the film-forming agent solution to undergo a cross-linking reaction. After the reaction is completed, the temperature is lowered to 42°C~48°C to obtain a fourth mixed solution. The third mixed solution is added to the fourth mixed solution, and the pH is adjusted to 5.5~7.5 to obtain a fifth mixed solution.

[0014] The antimicrobial peptide extract from the skin of Rana spinosa was added to the sodium hyaluronate solution and mixed evenly to obtain a sixth mixed solution.

[0015] The sixth mixed solution and the fifth mixed solution were mixed and then cooled to 35° C. to 38° C. to obtain the Rana spinosa skin antibacterial repair gel.

[0016] In a preferred embodiment of the present invention, in the film-forming agent solution, the ratio of film-forming agent to water is 1.5 g to 3 g: 85 ml to 100 ml.

[0017] The mass ratio of the film-forming agent in the film-forming agent solution to the trehalose in the second mixed solution is 1.5-3:5-7.

[0018] In the third mixed liquid, the ratio of butylene glycol, 1,2-hexanediol, phenoxyethanol and PEG-40 hydrogenated castor oil is 8ml~10ml: 1g~3g: 0.5ml~1.5ml: 0.3ml~0.8ml; 0.3ml~0.5ml.

[0019] The ratio of the film-forming agent in the film-forming agent solution to the butanediol in the third mixed solution is 1.5 g to 3 g: 8 ml to 10 ml.

[0020] In sodium hyaluronate solution, the ratio of sodium hyaluronate to water is 0.1g~0.3g:10ml~15ml The mass ratio of the film-forming agent in the film-forming agent solution to the sodium hyaluronate in the sodium hyaluronate solution is 1.5-3:0.1-0.3.

[0021] In a preferred embodiment of the present invention, in the first mixed solution, the ratio of glycerol to sodium carboxymethyl β-glucan is 8 ml to 10 ml: 0.2 g to 0.4 g. In the second mixed solution, the ratio of trehalose to disodium EDTA and water is 5 g to 7 g: 0.1 g to 0.2 g: 165 ml to 190 ml. The ratio of glycerol in the first mixed solution to trehalose in the second mixed solution is 8 ml to 10 ml: 5 g to 7 g.

[0022] In a preferred embodiment of the present invention, in the sixth mixed solution, the mass ratio of sodium hyaluronate to the antimicrobial peptide extract from the skin of Rana spinosa is 0.1-0.3:2-5.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses ultrasound-assisted acid hydrolysis of Rana spinosa skin to extract antimicrobial peptides, which reduces the use of solvents, reduces degradation of antimicrobial peptides during the extraction process, increases the extraction rate, and helps maintain the stability of the peptides. The antimicrobial peptides are freeze-dried to prevent thermal degradation of the peptides, which can easily denature at high temperatures and lose their three-dimensional structure and biological activity. After freeze-drying, moisture is removed, making it easier to store and maintaining its stability.

[0024] (2) The antimicrobial peptide extract from the skin of Rana spinosa prepared by the present invention has a higher antimicrobial peptide extraction rate of about 10%; a better antibacterial effect, with an inhibition zone diameter of about 5 mm; and a more stable antibacterial effect.

[0025] (3) The antibacterial repair gel of Rana spinosa skin prepared by the present invention has better skin feel, hydration, moisturizing properties and acne removal effect, and there is no sign of recurrence within a certain period of time.

[0026] (4) The method of the present invention is used to develop an antibacterial repair gel product for Rana spinosa skin, which has a better skin feel, more stable effect, and high cost performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 The standard curve of peptide concentration.

[0029] Figure 2 are the inhibition zones of antimicrobial peptides against Staphylococcus aureus and Propionibacterium acnes, among which ① is the inhibition zone of 20 μl antimicrobial peptide against Staphylococcus aureus, ② is the inhibition zone of 50 μl antimicrobial peptide against Staphylococcus aureus, ③ is the inhibition zone of 20 μl antimicrobial peptide against Propionibacterium acnes, and ④ is the inhibition zone of 50 μl antimicrobial peptide against Propionibacterium acnes.

[0030] Figure 3 These are the inhibition zones of the antibacterial repair gel against different bacteria, among which ① is Propionibacterium acnes and ② is Staphylococcus aureus. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] Example 1 Fresh frog skin was dried at 45°C for 24 hours to obtain dried frog skin. The dried skin was taken and an acetic acid solution with a concentration of 0.105g / ml was added at a material-liquid ratio of 1g:50ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 60 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a -80°C freeze dryer, concentrated under reduced pressure and freeze-dried to obtain a freeze-dried powder of the frog skin antimicrobial peptide extract.

[0033] Example 2 Fresh frog skin was dried at 45°C for 24 hours to obtain dried frog skin. The dried skin was taken and an acetic acid solution with a concentration of 0.1575g / ml was added at a material-liquid ratio of 1g:50ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 60 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a -80°C freeze dryer, concentrated under reduced pressure and freeze-dried to obtain a freeze-dried powder of the frog skin antimicrobial peptide extract.

[0034] Example 3 Fresh frog skin was dried at 45°C for 24 hours to obtain dried frog skin. The dried skin was taken and an acetic acid solution with a concentration of 0.21g / ml was added at a material-liquid ratio of 1g:50ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 60 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a -80°C freeze dryer, concentrated under reduced pressure and freeze-dried to obtain a freeze-dried powder of the frog skin antimicrobial peptide extract.

[0035] Table 1 The mass of freeze-dried powder obtained with different extract concentrations Lyophilized powder of an antimicrobial peptide extract from the skin of Rana spinosa was prepared according to the experimental methods of Examples 1 to 3. After five repetitions, the extraction yield results are shown in Table 1. The highest extraction yield was achieved when the acetic acid concentration was 0.105 g / ml. This is because the solubility, degree of ionization, and interaction of acetic acid with other substances in water may vary with concentration, thus affecting extraction efficiency. At a concentration of 0.105 g / ml, acetic acid exhibited optimal interaction with the target substance, or its partition coefficient in the solvent was optimal, resulting in the highest extraction yield.

[0036] Example 4 Fresh Rana spinosa skin was dried at 45°C for 24 hours to obtain Rana spinosa dry skin. The dry skin was taken and an acetic acid solution with a concentration of 0.105g / ml was added at a material-liquid ratio of 1g:50ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 90 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a -80°C freeze dryer, concentrated under reduced pressure and freeze-dried to obtain a freeze-dried powder of Rana spinosa skin antimicrobial peptide extract.

[0037] Example 5 Fresh Rana spinosa skin was dried at 45°C for 24 hours to obtain Rana spinosa dry skin. The dry skin was taken, and an acetic acid solution with a concentration of 0.105g / ml was added at a material-liquid ratio of 1g:50ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 120 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a -80°C freeze dryer, concentrated under reduced pressure and freeze-dried to obtain a freeze-dried powder of Rana spinosa skin antimicrobial peptide extract.

[0038] Table 2 The mass of freeze-dried powder obtained at different ultrasonic extraction times The results, as shown in Table 2, show that the highest extraction rate was achieved when the ultrasonic extraction time was 90 minutes. This is because the working principle of ultrasonic extraction technology relies primarily on the cavitation, thermal, and mechanical effects of ultrasound. These effects increase the frequency and speed of molecular motion, enhance the penetration of the solvent, and thus improve the leaching rate of the target component. At a 90-minute extraction time, these effects are fully realized, resulting in the highest extraction rate.

[0039] Example 6 Fresh frog skin was dried at 45°C for 24 hours to obtain dried frog skin. The dried skin was taken and an acetic acid solution with a concentration of 0.105g / ml was added at a material-liquid ratio of 1g:75ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 60 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a -80°C freeze dryer, concentrated under reduced pressure and freeze-dried to obtain a freeze-dried powder of the frog skin antimicrobial peptide extract.

[0040] Example 7 Fresh frog skin was dried at 45°C for 24 hours to obtain dried frog skin. The dried skin was taken and an acetic acid solution with a concentration of 0.105 g / ml was added at a material-liquid ratio of 1 g:100 ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500 W and 60°C for 60 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000 r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a -80°C freeze dryer, concentrated under reduced pressure and freeze-dried to obtain freeze-dried powder of frog skin antimicrobial peptide extract.

[0041] Table 3 Quality of freeze-dried powder obtained with different material-liquid ratios The results, shown in Table 3, show that the highest extraction yield was achieved when the solid-liquid ratio was 1g:75ml. This is because the solid-liquid ratio directly determines the extraction yield by influencing solubility, concentration gradient, wetting efficiency, and mass transfer dynamics. An appropriate solid-liquid ratio maximizes solute release, minimizes solvent waste, and thus improves the leaching rate of the target component. At a solid-liquid ratio of 1g:75ml, these interactions are fully realized, resulting in the highest extraction yield.

[0042] The characterization test of the antimicrobial peptide extract from the skin of Rana spinosa prepared by the present invention is as follows.

[0043] (1) Peptide content of antimicrobial peptide extract from Rana spinosa skin The antimicrobial peptide extract from the skin of Rana spinosa prepared in Example 2 was taken, and 1 ml of deionized water was measured to dissolve the antimicrobial peptide extract from the skin of Rana spinosa to prepare an antimicrobial peptide reconstitution solution with an antimicrobial peptide concentration of 1 mg / mL. The antimicrobial peptide content in the reconstitution solution was determined by the Coomassie Brilliant Blue method, and the antimicrobial peptide content in the reconstitution solution of the antimicrobial peptide extract from the skin of Rana spinosa was determined according to the polypeptide content standard curve.

[0044] Drawing of standard curve: Weigh 100 mg of Coomassie Brilliant Blue and dissolve it in 50 mL of 95% ethanol. Then add 100 mL of 85% phosphoric acid and dilute to 1 L with distilled water to obtain a mixed solution. Let the mixture stand for 2 hours, filter the mixture to remove the residue, and obtain the Coomassie Brilliant Blue solution. Place it in a brown bottle for later use.

[0045] Weigh 10 mg of bovine serum albumin and mix it evenly with 100 mL of distilled water to obtain a 0.1 mg / mL bovine serum albumin standard solution. Divide six stoppered test tubes, numbered 1 to 6 (i.e., groups), into which 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the bovine serum albumin standard solution are added, respectively. Fill all test tubes to 1 mL with distilled or deionized water (perform three replicates per group).

[0046] Use a pipette to draw 5mL of Coomassie Brilliant Blue solution and add it to each test tube in turn. Close the tube stopper tightly, invert it upside down 6 times, and let it stand for 2 minutes to obtain the standard solution (the measurement should be completed within 1 hour). Pour the standard solution into a 1cm quartz cuvette and perform colorimetry at a wavelength of 595nm. Draw a standard curve with the concentration of the polypeptide, i.e., bovine serum albumin, as the horizontal axis and the absorbance as the vertical axis. The standard curve is as shown below. Figure 1 shown.

[0047] (2) Antibacterial activity of antimicrobial peptide extracts from Rana spinosa skin 1 mL of the lyophilized powder of the antimicrobial peptide extract from the skin of Rana spinosa prepared in Example 2 (protein content 5-10 mg / mL) was weighed into a hydrolysis bottle, and 1 mL of sterile PBS solution was added to reconstitute the solution to prepare the reconstituted solution. The antibacterial activity of the reconstituted solution of the antimicrobial peptide extract from the skin of Rana spinosa was determined by the Oxford cup method.

[0048] Pour approximately 15 mL of LB solid medium into a Petri dish and allow the medium to solidify. Then, inoculate Staphylococcus aureus and Propionibacterium acnes grown to the logarithmic phase into the LB solid medium. Place an Oxford cup on the LB medium. Dilute the Rana spinosa skin extract to a concentration of 30 mg / mL. Add 20 μl and 50 μl of the sample solution to the wells of the Oxford cup, respectively. Sterile PBS buffer was used as a negative control. Cover the Petri dish and incubate overnight at 28°C. Observe the size and morphology of the inhibition zone. The antibacterial activity of the Rana spinosa skin antimicrobial peptide extract against Staphylococcus aureus (CMCC 26003) and Propionibacterium acnes (CMCC(B)65111) was tested.

[0049] Table 4 Inhibition zone diameters of antimicrobial peptides against Staphylococcus aureus and Propionibacterium acnes The results are as follows Figure 2 As shown in Table 4, Staphylococcus aureus is highly sensitive to the antimicrobial peptide extract from the skin of Rana spinosa, and Propionibacterium acnes is extremely sensitive to the antimicrobial peptide extract from the skin of Rana spinosa. The antimicrobial activity of the antimicrobial peptide extract from the skin of Rana spinosa is very strong.

[0050] (3) Minimum inhibitory concentration (MIC) of antimicrobial peptide extract from Rana spinosa skin Staphylococcus aureus and Propionibacterium acnes were cultured in LB medium to the logarithmic growth phase to obtain bacterial suspension, which was then diluted to 1×10 6CFU / mL; then take a 96-well culture plate, add 90 μL of bacterial solution to each well, weigh the corresponding grams of the antimicrobial peptide lyophilized powder prepared in Example 2, add 1 ml of sterile PBS to reconstitute and dilute to the following concentrations to obtain antimicrobial peptide solutions, and add 10 μL of antimicrobial peptide solutions of different concentrations to each well (concentration gradient is 250.00 μg / ml, 125.00 μg / ml, 62.50 μg / ml, 31.25 μg / ml, 15.63 μg / ml, 7.81 μg / ml, 3.91 μg / ml, 1.95 μg / ml, 0.98 μg / ml, 0.49 μg / ml, 0.24 μg / ml); add 100 μL / well of bacterial solution to the negative control, and add 100 μL / well of LB medium to the blank control; then culture each well at 37°C for 16 h, and measure 630 The MIC (Minimal Inhibitory Concentration) of the antimicrobial peptide extract from Rana spinosa skin was determined by measuring the absorbance at 40 nm. The MIC (Minimal Inhibitory Concentration) is defined as the lowest concentration of the antimicrobial peptide that inhibits bacterial growth by 100%.

[0051] Table 5 MIC values of antimicrobial peptides against Staphylococcus aureus and Propionibacterium acnes The results are shown in Table 5. The MIC of the antimicrobial peptide extract from the skin of R. spinosa was 7.81 μg / ml against Staphylococcus aureus and 3.91 μg / ml against Propionibacterium acnes.

[0052] Comparative Example 1 Fresh Rana spinosa skin was dried at 45°C for 24 hours to obtain Rana spinosa dry skin. The dry skin was taken, and an acetic acid solution with a concentration of 0.105g / ml was added at a material-liquid ratio of 1g:75ml and shaken to obtain a mixed solution. The mixed solution was then heated in a 60°C water bath and extracted for 90 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a 25°C oven, concentrated and dried to obtain a dry powder of Rana spinosa skin antimicrobial peptide extract.

[0053] Comparative Example 2 Fresh frog skin was dried at 45°C for 24 hours to obtain the dried frog skin. The dried skin was taken and an acetic acid solution with a concentration of 0.105g / ml was added at a material-liquid ratio of 1g:75ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 90 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a 25°C oven, concentrated and dried to obtain a dry powder of the antimicrobial peptide extract from the frog skin.

[0054] Comparative Example 3 Fresh frog skin was dried at 45°C for 24 hours to obtain the dried frog skin. The dried skin was taken and an acetic acid solution with a concentration of 0.105g / ml was added at a material-liquid ratio of 1g:75ml and shaken to obtain a mixed solution. The mixed solution was then extracted with ultrasound at 500W and 60°C for 90 minutes. After the extraction was completed, the mixture was filtered and the filtrate was collected. The filtrate was concentrated by rotary evaporation at 70°C for 20 minutes to obtain a crude extract. The crude extract was centrifuged at 6000r / min for 10 minutes, and the supernatant was collected. The supernatant was placed in a 25°C oven, concentrated and dried to obtain a dry powder of the antimicrobial peptide extract from the frog skin.

[0055] (4) Thermal stability test of antimicrobial peptide extract from Rana spinosa skin The stability of the antimicrobial peptide extract from the skin of Rana spinosa was tested by the punch method. 400 μL of Staphylococcus aureus suspension and Propionibacterium acnes were mixed with LB medium at 45-50°C to obtain bacterial suspension. The bacterial suspension was diluted to obtain a bacterial density of 1×10 6 CFU / mL of bacterial solution; 100 μL of bacterial solution with a bacterial density of 1×10 6 A bacterial solution containing 100 CFU / mL was spread onto LB solid medium (containing 1.3% agar) to obtain a culture medium containing bacteria. A 100 μg / mL antimicrobial peptide solution (50 μl) was prepared (100 μg of the lyophilized powder of the antimicrobial peptide extract from the skin of Rana spinosa prepared in Example 2 was dissolved in 1 ml of sterile PBS) and incubated at 40°C, 60°C, 80°C, and 100°C. Samples were taken at 15 and 30 minutes and added to the wells in the culture medium containing bacteria. After incubation at 37°C for 24 hours, the diameter of the inhibition zone in the culture dish was measured. The average value was obtained after four replicates. Antimicrobial peptides not incubated (20°C) were used as controls. The results are shown in Table 6.

[0056] Table 6 Inhibition zone diameters of antimicrobial peptides against Staphylococcus aureus and Propionibacterium acnes at different temperatures The results are shown in Table 6. The difference in the diameter of the inhibition zone was observed to be ±0.6 mm (<1 mm), indicating that the antimicrobial peptide prepared by the present invention has strong thermal stability.

[0057] Table 7 Antibacterial data of different samples The results are shown in Table 7. After incubation at 36°C for 24 hours, a comparison of Comparative Examples 1 and 2 shows that the ultrasonic extraction method produces a superior antibacterial effect for the dry powder of the antimicrobial peptide extract from the skin of R. spinosa. This is because ultrasonic extraction generates high-frequency vibrations, which are more effective in disrupting cell structure and promoting the release of antimicrobial peptides. Therefore, the ultrasonic extraction method produces a superior antibacterial effect for the dry powder of the antimicrobial peptide extract from the skin of R. spinosa. It should be noted that the antibacterial data in Table 7 were obtained by incubating 50 μl of a 100 μg / ml antimicrobial peptide solution at room temperature (25°C) for 15 minutes, then adding it to a culture medium containing bacteria and incubating it at 37°C for 24 hours. The diameter of the inhibition zone in the culture dish was measured and averaged after four repetitions. Other conditions were the same as those for the thermal stability test.

[0058] Comparing Comparative Example 1 with Comparative Example 3, where freeze-drying was used, it can be seen that the antibacterial efficacy of the Rana spinosa skin antimicrobial peptide extract powder is enhanced after freeze-drying. This is because freeze-drying is performed at low temperatures (typically below -50°C), which helps prevent thermal degradation of the peptides. Peptides are easily denatured at high temperatures, losing their three-dimensional structure and biological activity. Conventional drying methods may require higher temperatures, which can lead to chemical modification or cross-linking of the peptides, but freeze-drying avoids these problems. However, the antibacterial efficacy of Comparative Example 3 is still lower than that of Example 4, indicating that the Rana spinosa skin antimicrobial peptide extract powder prepared by the extraction method of the present invention possesses excellent antibacterial efficacy. This demonstrates that the extraction method of the present invention utilizes multiple synergistic optimization effects to achieve excellent antibacterial efficacy and stability in the Rana spinosa skin antimicrobial peptide extract powder. First, ultrasonic extraction is used in the extraction process to precisely and efficiently disrupt the complex cell structure of Rana spinosa skin, promoting rapid and comprehensive release of the antimicrobial peptides from the cell interior, significantly improving extraction efficiency while preserving the original active structure of the antimicrobial peptides to the greatest extent possible, resulting in enhanced antibacterial properties. Then, freeze-drying technology is used in the drying stage, and low-temperature operation can effectively prevent thermal degradation of the peptide. Compared with ordinary drying methods, it avoids problems such as peptide denaturation, chemical modification or cross-linking caused by high temperature, ensuring that the three-dimensional structure and biological activity of the antimicrobial peptide are not damaged. Finally, in terms of stability, the data of multiple repeated experiments are highly consistent. At different times and by different operators, the inhibition zone diameter and inhibition strength data of the two bacteria in Example 4 remain almost constant. Even if the antimicrobial peptide extraction sample is between the experimental temperature of 25°C and 100°C, its antibacterial effect still fluctuates only slightly within the range, and based on the inevitable subtle differences in experimental operations, its antibacterial effect remains stable.

[0059] Next, the Rana spinosa skin antibacterial repair gel was prepared using the extracted antibacterial peptide extract. During the preparation, glycerol and sodium carboxymethyl β-glucan were evenly mixed to obtain a first mixed solution; trehalose, disodium ethylenediaminetetraacetic acid and water were evenly mixed to obtain a second mixed solution; and butylene glycol, 1,2-hexanediol, phenoxyethanol and PEG-40 hydrogenated castor oil were evenly mixed to obtain a third mixed solution.

[0060] At 80°C–90°C, the second mixed solution and the first mixed solution containing sodium carboxymethyl β-glucan and disodium EDTA are added to the film-forming agent solution to undergo a cross-linking reaction, forming a more stable complex. High temperatures may promote the interaction between glycerin and the film-forming agent, enhancing film-forming properties. The temperature is then lowered to 42°C–48°C to produce a fourth mixed solution. The third mixed solution is then added to the fourth mixed solution, and the pH is adjusted to 5.5–7.5 to alter the charge state of sodium carboxymethyl β-glucan, thereby enhancing its interaction with other components. Components in the third mixed solution, such as phenoxyethanol, may exert their antimicrobial properties, improving the antiseptic properties of the final product, resulting in a fifth mixed solution.

[0061] The antimicrobial peptide extract from the skin of Rana spinosa was added to the sodium hyaluronate solution and mixed evenly to obtain a sixth mixed solution.

[0062] The sixth mixed solution is then allowed to interact with the fifth mixed solution, forming a more stable complex between sodium hyaluronate and sodium carboxymethyl β-glucan. The interaction between the Rana spinosa skin antimicrobial peptide extract and the other components facilitates its uniform distribution throughout the final product, exerting its antibacterial properties. The mixture is then cooled to 35°C-38°C to obtain the Rana spinosa skin antimicrobial repair gel.

[0063] The acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer used in the present invention was purchased from Guangzhou Ruishi Biotechnology Co., Ltd.

[0064] Application Example 1 Step 1: Slowly disperse 3 g of acrylic acid (esters) / C10-30 alkyl acrylate crosslinked polymer into 90.0 ml of distilled water at 22° C., stir for 20 minutes until it is completely submerged, then stir and heat to 80° C., then sequentially add 184.6 ml of distilled water, 6.0 g of trehalose, and 0.15 g of disodium edetate. After mixing evenly, add a mixed solution I composed of 9.0 ml of glycerol and 0.3 g of sodium carboxymethyl β-glucan, and maintain the mixture at a constant temperature of 85° C. to fully dissolve. Finally, stir and cool the mixture to 45° C. to obtain a total solution I with a volume of 400 ml, wherein the concentration of acrylic acid (esters) / C10-30 alkyl acrylate crosslinked polymer in the total solution I is 0.75 g / ml.

[0065] Step 2: Mix 9.0 ml of butanediol, 2.1 g of 1,2-hexanediol, 0.75 ml of phenoxyethanol, and 0.45 ml of PEG-40 hydrogenated castor oil to obtain a mixed solution II. Heat the mixed solution II to 45° C. and add it to solution I. Stir evenly and adjust the pH to 6.5 with a 10 mg / ml aqueous sodium hydroxide solution to obtain solution II.

[0066] Step 3: Slowly disperse 0.12 g of sodium hyaluronate into 12 ml of distilled water. After it is completely dissolved, add 3 g of the freeze-dried powder of the antimicrobial peptide extract of Rana spinosa skin prepared in Example 2 to prepare a mixed solution III. Then, add the mixed solution III to solution II, stir evenly, and cool to 38° C. to obtain the Rana spinosa skin antibacterial repair gel.

[0067] Application Example 2 Step 1: Slowly disperse 2 g of acrylic acid (esters) / C10-30 alkyl acrylate crosslinked polymer into 90 ml of distilled water at 22° C., stir for 20 minutes until it is completely submerged, then stir and heat to 90° C., then sequentially add 165 ml of distilled water, 6.0 g of trehalose, and 0.2 g of disodium edetate. After mixing evenly, add a mixed solution I composed of 9.0 ml of glycerol and 0.2 g of sodium carboxymethyl β-glucan, and fully dissolve at a constant temperature of 85° C. Finally, stir and cool the temperature to 45° C. to obtain a total solution I with a volume of 400 ml, wherein the concentration of acrylic acid (esters) / C10-30 alkyl acrylate crosslinked polymer in the total solution I is 0.75 g / ml.

[0068] Step 2: Mix 9 ml of butanediol, 1 g of 1,2-hexanediol, 0.75 ml of phenoxyethanol, and 0.45 ml of PEG-40 hydrogenated castor oil to obtain a mixed solution II. Heat the mixed solution II to 42° C. and add it to solution I. Stir evenly and adjust the pH to 5.5 with a 10 mg / ml aqueous sodium hydroxide solution to obtain solution II.

[0069] Step 3: Slowly disperse 0.3 g of sodium hyaluronate into 12 ml of distilled water. After it is completely dissolved, add 5 g of the freeze-dried powder of the antimicrobial peptide extract of Rana spinosa skin prepared in Example 1 to prepare a mixed solution III. Then, add the mixed solution III to the solution II, stir evenly, and cool to 38° C. to obtain the Rana spinosa skin antibacterial repair gel.

[0070] Application Example 3 Step 1: Slowly disperse 1.5 g of acrylic acid (esters) / C10-30 alkyl acrylate crosslinked polymer into 90 ml of distilled water at 22° C., stir for 20 minutes until it is completely submerged, then stir and heat to 80° C., then sequentially add 190 ml of distilled water, 6.0 g of trehalose, and 0.1 g of disodium edetate. After mixing evenly, add a mixed solution I composed of 9.0 ml of glycerol and 0.4 g of sodium carboxymethyl β-glucan, and maintain the mixture at a constant temperature of 85° C. to fully dissolve. Finally, stir and cool the mixture to 48° C. to obtain a total solution I with a volume of 400 ml, wherein the concentration of acrylic acid (esters) / C10-30 alkyl acrylate crosslinked polymer in the total solution I is 0.75 g / ml.

[0071] Step 2: Mix 9 ml of butanediol, 3 g of 1,2-hexanediol, 0.75 ml of phenoxyethanol, and 0.45 ml of PEG-40 hydrogenated castor oil to obtain a mixed solution II. Heat the mixed solution II to 42° C. and add it to solution I. Stir evenly and adjust the pH to 7.5 with a 10 mg / ml aqueous sodium hydroxide solution to obtain solution II.

[0072] Step 3: Slowly disperse 0.16 g of sodium hyaluronate into 19 ml of distilled water. After it is completely dissolved, add 6.7 g of the freeze-dried powder of the antimicrobial peptide extract of Rana spinosa skin prepared in Example 1 to prepare a mixed solution III. Then, add the mixed solution III to solution II, stir evenly, and cool to 38° C. to obtain the Rana spinosa skin antibacterial repair gel.

[0073] The antibacterial activity was determined using the Rana spinosa skin antibacterial repair gel prepared in Example 1 as an example.

[0074] Pour approximately 15 mL of LB solid medium into a Petri dish and allow the medium to solidify. Inoculate Staphylococcus aureus and Propionibacterium acnes grown to the logarithmic phase into the LB solid medium. Place an Oxford cup on the medium. Add 50 μl of the Rana spinosa skin antibacterial repair gel to the wells of the Oxford cup. Use sterile PBS buffer as a negative control. Cover the Petri dish and incubate overnight at 28°C. Observe the size and morphology of the inhibition zone. The antibacterial activity of the Rana spinosa skin antibacterial repair gel against Staphylococcus aureus and Propionibacterium acnes was tested.

[0075] Table 8 Inhibition zone diameters of antibacterial repair gel against Staphylococcus aureus and Propionibacterium acnes like Figure 3 As shown in Table 8, Staphylococcus aureus is highly sensitive to the Rana spinosa skin antibacterial repair gel, and Propionibacterium acnes is extremely sensitive to the Rana spinosa skin antibacterial repair gel. The Rana spinosa skin antibacterial repair gel has extremely strong antibacterial activity.

[0076] Twenty-four trial subjects aged 14-45 were selected. When the trial subjects first developed acne, the antibacterial repair gel prepared in Example 1 was used for one week, and the acne was significantly improved. At the same time, signs of acne recurrence were observed. The Rana spinosa skin antibacterial repair gel was comprehensively evaluated in terms of skin feel, hydration, moisturizing properties, and acne removal effect. The results are shown in Table 9.

[0077] It should be noted that skin feel, hydration, moisturizing properties and acne-removing effects are objectively scored based on the user experience and divided into three levels using a 10-point system: 1-4 points, 5-7 points and 8-10 points, among which 1-4 points indicate poor, 5-7 points indicate good, and 8-10 points indicate excellent.

[0078] The time for effective acne removal is divided into 10h~20h, 21h~40h, 41h~60h and 61h~80h according to the survey results. Among them, 10h~20h indicates that acne removal is effective in the initial infection stage of acne, 21h~40h indicates that acne removal is effective in the growth stage of acne, 41h~60h indicates that acne removal is effective in the mature stage of acne, and 61h~80h indicates that acne removal is effective in the stable stage of acne.

[0079] It should be noted that in Table 9, when the number of raters is greater than 1, the evaluation score is the average value; when the number of raters is 1, the evaluation score is the specific value.

[0080] Table 9 Experience of different groups using antibacterial repair gel During the trial period, 80% of the trial users felt that their acne had faded or improved to a certain extent, and there was no sign of recurrence for a certain period of time; and 65% of the trial users felt that their skin felt better and more hydrated, but the moisturizing effect needed further improvement and enhancement.

[0081] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0082] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing an antimicrobial peptide extract from the skin of Rana spinosa, characterized in that: The following steps are involved: The dried skin of Rana spinosa was ultrasonically extracted at 60℃~70℃ using acetic acid solution as the extraction solution, and post-processing was performed after the extraction was completed. After the post-processing was completed, freeze-drying was performed to obtain the antimicrobial peptide extract from the skin of Rana spinosa.

2. The method for preparing the antimicrobial peptide extract from the skin of Rana spinosa according to claim 1, wherein: The ratio of dried skin of Rana spinulosa to acetic acid solution is 1g:50ml~100ml, and the concentration of acetic acid solution is 0.105g / ml~0.21g / ml.

3. The method for preparing the antimicrobial peptide extract from the skin of Rana spinosa according to claim 1, wherein: The extraction time is 60min~120min.

4. The method for preparing the antimicrobial peptide extract from the skin of Rana spinosa according to claim 1, wherein: The ultrasonic power is 500W~600W.

5. An antimicrobial peptide extract from the skin of Rana spinosa obtained by the preparation method according to any one of claims 1 to 4.

6. A Rana spinosa skin antibacterial repair gel, characterized in that: The invention comprises the Rana spinosa skin antimicrobial peptide extract according to claim 5.

7. A method for preparing the Rana spinosa skin antibacterial repair gel according to claim 6, characterized in that: The following steps are involved: Glycerin and sodium carboxymethyl β-glucan are mixed evenly to obtain a first mixed solution; trehalose, disodium edetate and water are mixed evenly to obtain a second mixed solution; butanediol, 1,2-hexanediol, phenoxyethanol and PEG-40 hydrogenated castor oil are mixed evenly to obtain a third mixed solution; adding the second mixed solution and the first mixed solution to the film-forming agent solution at 80° C. to 90° C. to allow sodium carboxymethyl β-glucan to crosslink with disodium edetate. After the reaction is complete, lowering the temperature to 42° C. to 48° C. to obtain a fourth mixed solution; adding the third mixed solution to the fourth mixed solution and adjusting the pH to 5.5 to 7.5 to obtain a fifth mixed solution; Add the antimicrobial peptide extract from the skin of Rana spinosa to the sodium hyaluronate solution and mix evenly to obtain a sixth mixed solution; The sixth mixed solution and the fifth mixed solution were mixed and then cooled to 35° C. to 38° C. to obtain the Rana spinosa skin antibacterial repair gel.

8. The method for preparing the antibacterial repair gel of Rana spinosa skin according to claim 7, characterized in that: In the film-forming agent solution, the ratio of film-forming agent to water is 1g~2g:60ml; The mass ratio of the film-forming agent in the film-forming agent solution to the trehalose in the second mixed solution is 1-2:4; In the third mixed solution, the ratio of butanediol to 1,2-hexanediol is 9ml:1g~3g; The ratio of 1,2-hexanediol and phenoxyethanol is 100g~300g:75ml; The ratio of 1,2-hexanediol and PEG-40 hydrogenated castor oil is 100g~300g:45ml; The ratio of the film-forming agent in the film-forming agent solution to the butanediol in the third mixed solution is 1 g to 2 g: 6 ml; In the sodium hyaluronate solution, the ratio of sodium hyaluronate to water is 0.1g~0.3g:12ml; The mass ratio of the film-forming agent in the film-forming agent solution to the sodium hyaluronate in the sodium hyaluronate solution is 12.5-25:

1.

9. The method for preparing the antibacterial repair gel of Rana spinosa skin according to claim 7, characterized in that: In the first mixed solution, the ratio of glycerol to sodium carboxymethyl β-glucan is 45ml: 1g~2g; In the second mixed solution, the ratio of trehalose to disodium edetate is 60g:1g-2g; The ratio of trehalose to water is 6g:165ml~190ml; The ratio of glycerol in the first mixed solution to disodium edetate in the second mixed solution is 90 ml: 1 g to 2 g.

10. The method for preparing the antibacterial repair gel of Rana spinosa skin according to claim 7, characterized in that: In the sixth mixed solution, the mass ratio of sodium hyaluronate to the antimicrobial peptide extract from the skin of Rana spinosa is 12:200-500.

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