Preparation method of crocodile skin collagen peptide and application of composition of crocodile skin collagen peptide in antisepsis, anti-inflammation and skin repair

Abstract

The invention discloses a preparation method of crocodile skin collagen peptide and application of a composition of the crocodile skin collagen peptide in antisepsis, anti-inflammation and skin repair. The crocodile skin collagen peptide provided by the invention is composed of 17 peptide fragments with the molecular weight of 700-1800 Da. The composition obtained by compounding crocodile skin collagen peptide, mandelic acid, lactobionic acid, asiaticoside and nicotinamide has multiple functions of antisepsis, anti-inflammation, skin repair and the like through multi-target point synergism, and the crocodile skin collagen peptide dominates oxidation resistance and skin repair and delays skin aging; mandelic acid is used for mildly conditioning cutin, effectively dredging pores and inhibiting growth and reproduction of acne-related flora; lactobionic acid promotes epidermis renewal, makes the skin moist and delicate, and reduces fine wrinkles; the asiaticoside is used for relieving, diminishing inflammation and relieving irritation reactions such as skin redness and swelling; nicotinamide can regulate sebum secretion, block melanin migration and brighten the skin color. The composition can effectively deal with acnes and complications, and solves the problems that an existing product is high in irritation, easy to leave marks and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a preparation method of crocodile skin collagen peptide and application of a composition thereof in antibacterial anti-inflammatory and skin repair. BACKGROUND

[0002] Acne, also known as "acne vulgaris", "dark spots" or "pimples", is a relatively common chronic inflammatory skin disease, which often occurs on the cheeks, forehead and lower jaw, and mainly occurs in adolescents. Its pathogenesis is complex, mainly involving four key links: excessive secretion of lipids by sebaceous glands, abnormal keratinization of sebaceous duct, excessive proliferation of microorganisms such as Propionibacterium acnes and inflammatory reactions induced by them. Although there are many acne treatment products on the market, there are obvious limitations, and it is difficult to achieve satisfactory efficacy. For example, although retinoids can effectively regulate keratinization, they have a slow onset and strong irritation, which can easily cause skin dryness, desquamation and photosensitivity; the bactericidal effect of benzoyl peroxide and other oxidizing agents is significant, but their strong oxidizing property often causes strong skin irritation and damages the skin barrier function, which may even exacerbate skin sensitivity and inflammation. In addition, most existing functional skin care products have low active ingredient concentration or single function, and cannot simultaneously intervene in multiple pathogenic links of acne, resulting in poor efficacy for moderate to severe acne. Therefore, there is an urgent need in the market for a new type of composite active composition product that can act on multiple targets, is efficient and mild, to achieve comprehensive prevention and treatment of acne and skin repair.

[0003] The existing research on acne treatment products containing multiple active ingredients has shown that each ingredient has certain potential for acne treatment, but acne has a complex pathogenesis and needs to be treated through multiple pathways. However, simple combination of multiple ingredients with different mechanisms often faces many challenges, such as poor compatibility between ingredients, conflict in physical and chemical properties (such as pH difference), mutual antagonism of efficacy or superposition of irritation, etc. Therefore, it is of great significance and market urgency to develop a new type of composite composition that is efficient, multi-effect synergistic and mild and low-irritating.

[0004] Crocodiles are rare biological resources, and their tissues are rich in functional polypeptides with unique structures. These polypeptides have been proven to have excellent properties of strong antioxidant and promotion of tissue repair. Developed countries have established industrialized preparation systems for crocodile active ingredients based on advanced biological extraction technology, and successfully applied them in the fields of high-end medical dressings and wound repair preparations. China's crocodile large-scale breeding industry has become mature, but the current development level is still mainly concentrated in the primary consumption fields of catering, leather and tourism, and there is still a significant technical gap in the extraction and application of high-value biological active ingredients, resulting in a huge waste of valuable biological resources.

[0005] Collagen peptides are a mixture of low molecular weight peptides generated by controlled hydrolysis of collagen. Due to their excellent absorbability, biocompatibility and diverse physiological activities (such as antibacterial, moisturizing, repair, antioxidant and anti-inflammatory), collagen peptides have become one of the core raw materials in the health industry. Currently, commercial collagen peptides are mainly derived from animal tissues such as pigs, cows and fish, and few other sources of collagen resources are applied. In contrast, alligator skin is a new source of high-quality collagen with great development potential. Its advantages are obvious: first, the collagen content of alligator is significantly higher than that of traditional sources such as pigs and cows; second, alligators are far away from the human food chain, and the risk of allergy is extremely low. Most importantly, thanks to the strong innate immune system of alligators, the collagen peptides from their skin may contain unique active fragments, exhibiting better antioxidant and tissue repair biological activities than traditional sources, which provides a new opportunity and scientific basis for the development of a new generation of high-end functional cosmetics.

[0006] Under this background, China's alligator industry needs to be transformed and upgraded to deep processing. Establishing an extraction and transformation technology system centered on the biological active ingredients of alligators and deeply exploring the application value of alligators in the fields of medicine, cosmetics, health and other fields is a strategic path for China to break through the industrial bottleneck and realize resource value-added. SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the problems of strong irritation, easy damage to the skin, single function and easy recurrence of the existing acne-removing and anti-inflammatory products for treating acne, as well as the insufficient development and application of alligator resources. The present application provides a preparation method of alligator skin collagen peptides and the application of the composition thereof in antibacterial and anti-inflammatory skin repair.

[0008] The first object of the present application is to provide an alligator skin collagen peptide.

[0009] The second object of the present application is to provide a preparation method of alligator skin collagen peptide.

[0010] The third object of the present application is to provide a composition containing alligator skin collagen peptide.

[0011] The fourth object of the present application is to provide a preparation method and application of the composition containing alligator skin collagen peptide.

[0012] The fifth object of the present application is to provide a product.

[0013] The above objects of the present application are achieved by the following technical solutions: The application provides a crocodile skin collagen peptide, which comprises the following peptide segments: ARLLLAPAK, CCKAQEPEACFK, ELLENEFHQILK, FKNGMLHGQK, KQPKPLK, KSFDVVKHK, LESDVSTQMEYCR, LVTDLTK, NEEQSGDGSR, PHHSPLVLKR, QQFLINLVKQK, SLLPVTK, TILGEQFPKLAK, VGGVGVPAAPSITAVTVNK, VSDALSGDR, WKIAFDKGR and YETELALR.

[0014] The application is prepared by extracting crocodile skin, and a crocodile skin collagen peptide is obtained, which comprises 17 peptide segments, the molecular weight of the peptide segments is about 700-1800 Da, the crocodile skin collagen peptide has strong antioxidant capacity, high stability and good biological safety. The chemical composition of the crocodile skin collagen peptide is analyzed by using an ultra-high performance liquid chromatography-Orbitrap high-resolution mass spectrometry technology, and combined with mz-Cloud database retrieval, 20 compounds are identified in the crocodile skin collagen peptide, and the main components include small molecule peptides, amino acids and nitrogen-containing metabolites, and the five components with the highest content are glycyl proline (19.84%), DL-arginine (17.25%), choline (8.43%), glycyl-L-leucine (8.28%) and diethyl phosphate (9.64%), and in addition, prolyl leucine, alpha-aspartyl phenylalanine, cyclo (leucyl prolyl) and other small molecule peptides are detected, which shows that the crocodile skin collagen peptide is rich in various active substances and short-chain peptides. The crocodile skin collagen peptide provided by the application has high purity and activity, and can be better applied to the preparation of subsequent drugs or cosmetic products.

[0015] The application provides a preparation method of the crocodile skin collagen peptide, which comprises the following steps. S1. preparing crocodile skin collagen protein: washing, crushing, removing impurities and defatting the crocodile skin, then purifying, dialyzing to neutral, freeze-drying to obtain crocodile skin collagen protein powder for standby; S2. dissolving appropriate crocodile skin collagen protein powder, then performing enzymolysis by using a protease, inactivating and centrifuging after the enzymolysis, then performing ultrafiltration and desalting treatment, and then purifying to finally obtain the crocodile skin collagen peptide.

[0016] Preferably, the protease for the enzymolysis is one or more of alkaline protease, neutral protease, bromelain, pepsin and trypsin.

[0017] More preferably, the protease is alkaline protease.

[0018] Preferably, the enzymolysis conditions are: temperature 40~55℃, enzyme addition amount 15000~20000 U / g, enzymolysis 2~4 h.

[0019] The application also provides a specific preferred preparation method step of the alligator skin collagen peptide: S1. Preparation of alligator skin collagen: (1) Pretreatment: the thawed alligator skin is washed with clean water to remove the surface mud, bloodstains, scales and residual tissues, cut into small pieces and then crushed in a crusher; (2) Impurity removal and degreasing: the cut alligator skin is added into a 7.5 g / 100 mL NaCl solution, the ratio of alligator skin to NaCl solution is 1:10 (m / V), stirred at 4℃ for 12 h and then washed with distilled water. 10% n-butanol solution (volume ratio 1:10) is added, stirred at 4℃ for 24 h for degreasing, the solution is replaced every 8 h, and finally the alligator skin is washed with distilled water; (3) Extraction and purification: the pretreated alligator skin is placed in 20 times the volume of 0.5 mol / L acetic acid for extraction for 24 h, and stirred from time to time, then the viscous gelatinous liquid is separated, the residue is extracted under the same conditions for 24 h, and the extraction liquids of the two times are combined, a certain amount of NaCl solution is added to a final concentration of 0.9 mol / L overnight, centrifuged at 4℃ and 10000xg for 15 min, the supernatant is discarded, 10 times the volume of 0.5 mol / L acetic acid is added for dissolution, and the salting-out and centrifugation operations are repeated once. Dialsed in 10 times the volume of 0.1 mol / L acetic acid solution for 12 h, the solution is replaced every 4 h, then dialysed with distilled water to neutral, and freeze-dried for standby. At this time, the alligator skin collagen powder is obtained; S2. The appropriate alligator skin collagen powder is weighed, added into ultrapure water at a solid-liquid ratio of 50:1 (mg / mL), fully dissolved by stirring in a water bath at an appropriate temperature, and kept at the optimal enzymolysis temperature for 20 min. The alkaline protease is taken out and restored to room temperature, the pH is adjusted to the optimal reaction pH of the protease by using 1 mol / L or NaOH, the enzyme addition amount is 16000 U / g, and the enzymolysis is performed for 4 h. After inactivation at 95℃ for 10 min, centrifugation, ultrafiltration by a 3 kDa ultrafiltration tube, desalting by a desalting column, and purification by a dextran gel column-50, the alligator skin collagen peptide is prepared.

[0020] The application provides the use of the above-mentioned alligator skin collagen peptide in the preparation of an antioxidant product.

[0021] The application provides an alligator skin collagen peptide-containing composition, which contains the following ingredients in parts by weight: 0.1-10 parts of alligator skin collagen peptide, 0.5-15 parts of mandelic acid, 1-20 parts of lactobionic acid, 0.1-5 parts of asiaticoside and 2-10 parts of nicotinamide.

[0022] The application is prepared by compounding alligator skin collagen peptide, mandelic acid, lactobionic acid, asiaticoside and nicotinamide, and synergistic effects are achieved among the components; the alligator skin collagen peptide dominates antioxidant and skin repair, and delays skin aging; the mandelic acid effectively dredges pores by gently conditioning keratin, and inhibits the growth and reproduction of acne-related bacteria; the lactobionic acid promotes epidermal turnover, makes the skin moist and delicate, and reduces fine lines; the asiaticoside focuses on soothing and anti-inflammation, and reduces skin irritation such as redness; and the nicotinamide adjusts sebum secretion, blocks melanin migration, and brightens skin color. The composition has multiple functions such as antibacterial and anti-inflammatory, skin repair, etc. through multi-target synergistic effect, effectively copes with acne and complications, and prevents scar formation. The composition provided by the application can effectively improve the skin microenvironment, reduce the recurrence of acne and prevent scar formation, promote skin self-repair, improve overall skin quality, and is suitable for various skin types without side effects.

[0023] Preferably, the composition contains the following ingredients in parts by weight: 1-10 parts of alligator skin collagen peptide, 1-10 parts of mandelic acid, 5-15 parts of lactobionic acid, 1-5 parts of asiaticoside, and 2-10 parts of nicotinamide.

[0024] The application further provides a preparation method of the above composition, and the alligator skin collagen peptide, the mandelic acid, the lactobionic acid, the asiaticoside and the nicotinamide are weighed according to the proportion, mixed with a solvent at room temperature, and stirred at 200-300 r / min for 20-30 minutes until completely homogenized to obtain the composition.

[0025] Preferably, the solvent is pure water or ethanol.

[0026] The application provides application of the above composition in preparation of products for antibacterial and anti-inflammatory skin repair.

[0027] Preferably, the product is a medicine or a cosmetic.

[0028] Preferably, the product is a cosmetic; preferably, the product is an essence, a lotion, a mask, a gel, a cream or the like.

[0029] The application provides application of the above composition in preparation of a medicine for treating acne, folliculitis, seborrhea and acne marks.

[0030] In addition, the application further provides a product containing alligator skin collagen peptide or the above composition.

[0031] The application has the following beneficial effects: The application prepares a crocodile skin collagen peptide, which comprises 17 peptide segments, the molecular weight of the peptide segments is about 700-1800 Da, and the crocodile skin collagen peptide has strong antioxidant capacity. 20 compounds are identified in the crocodile skin collagen peptide, and the main components include small molecule peptides, amino acids and nitrogen-containing metabolites, and the five components with the highest content are: glycolproline (19.84%), DL-arginine (17.25%), choline (8.43%), glycol-L-leucine (8.28%), diethyl phosphate (9.64%), etc. In addition, prolyl leucine, alpha-aspartyl phenylalanine, cyclo (leucyl prolyl) and other small molecule peptides are detected, which shows that the crocodile skin collagen peptide prepared by the application is rich in various active substances and short chain peptides.

[0032] Meanwhile, the crocodile skin collagen peptide provided by the application, as a natural antioxidant raw material, has an unexpected effect of "1+1>2" in antioxidant, skin repair and reducing irritation after being compounded with different active ingredients. By scientifically compounding the five active ingredients of crocodile skin collagen peptide, mandelic acid, lactobionic acid, asiaticoside and nicotinamide, the excellent effect of multi-pathway synergy and comprehensive conditioning is exhibited, and the limitations of the prior art in single efficacy, high irritation and easy recurrence are significantly overcome.

[0033] Specifically, the crocodile skin collagen peptide as the core functional ingredient can effectively delay skin aging and repair skin activity due to its strong antioxidant properties; mandelic acid as a lipophilic fruit acid can gently unblock clogged pores, dissolve excessive accumulated keratin, and significantly improve the penetration efficiency of other active ingredients; lactobionic acid focuses on moisturizing and barrier repair, regulates the skin surface microecology, and accelerates the metabolism of old and worn-out keratin, thereby effectively lightening pigment type acne marks; asiaticoside further strengthens the anti-inflammatory effect by inhibiting the release of key inflammatory factors; nicotinamide regulates sebum secretion from the source and inhibits the transfer of melanin to keratinocytes, achieving the dual effect of oil control and acne mark lightening. After compounding the components in a specific ratio, the skin physiological gains of antibacterial and anti-inflammatory skin repair are synergistically achieved under the premise of ensuring low irritation and low sensitization. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The figure is the antioxidant activity effect diagram of each group of enzymes (in the figure, a is DPPH clearance rate; b is ABTS clearance rate).

[0035] Figure 2 The figure is the total ion flow chromatogram of crocodile skin collagen peptide (in the figure, a is positive ion mode; b is negative ion mode).

[0036] Figure 3The results of the changes in the auricle skin of each group of mice (a: blank group; b: model group; c: positive group; d: Example 2 group; e: Comparative Example 4).

[0037] Figure 4 The auricle skin of each group of mice (a: blank group; b: model group; c: positive group; d: Example 2 group; e: Comparative Example 4). DETAILED DESCRIPTION

[0038] The present application is further illustrated by the following description in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Example 1 Preparation and identification analysis of alligator skin collagen polypeptide 1. Preparation of alligator skin collagen S1. Pretreatment: The thawed alligator skin was washed with clean water to remove surface dirt, bloodstains, scales and residual tissue, cut into small pieces and then pulverized in a pulverizer.

[0041] S2. Defatting: The cut alligator skin was added to a 7.5 g / 100 mL NaCl solution, with a ratio of alligator skin to NaCl solution of 1:10 (m / V), stirred at 4 ℃ for 12 h, and washed with distilled water. 10% n-butanol solution (volume ratio 1:10) was added, and the alligator skin was defatted by stirring at 4 ℃ for 24 h, with the solution being replaced every 8 h. Finally, the alligator skin was washed with distilled water.

[0042] S3. Extraction and purification: The pretreated alligator skin was placed in 20 times the volume of 0.5 mol / L acetic acid for extraction for 24 h, with occasional stirring, then the viscous gel-like liquid was separated, and the residue was extracted under the same conditions for 24 h. The combined extract was added with a certain amount of NaCl solution to a final concentration of 0.9 mol / L overnight, and then centrifuged at 4 ℃ and 10000xg for 15 min. The supernatant was discarded, and 10 times the volume of 0.5 mol / L acetic acid was added for dissolution. The salting-out and centrifugation operations were repeated once. The extract was dialyzed in 10 times the volume of 0.1 mol / L acetic acid solution for 12 h, with the solution being replaced every 4 h, and then dialyzed to neutral with distilled water. The freeze-dried alligator skin collagen powder was obtained for use.

[0043] 2. Screening of protease for enzymatic hydrolysis Because of the different action sites of different protease species on peptide bonds, many studies have shown that the antioxidant activities of polypeptide products obtained by hydrolysis of different proteins by different proteases differ greatly. In this embodiment, six commonly used proteases were selected as the hydrolysis proteases for the preparation of alligator skin collagen peptide. Each enzyme was used to hydrolyze alligator skin collagen under the recommended optimal hydrolysis conditions.

[0044] An appropriate amount of alligator skin collagen protein powder was weighed, and ultrapure water was added at a solid-liquid ratio of 50:1 (mg / mL). The mixture was stirred in a water bath at an appropriate temperature until it was fully dissolved. Different proteases (alkaline protease, papain, bromelain, pepsin, and trypsin) were used for enzyme hydrolysis. After reaching the optimal enzyme hydrolysis temperature of each enzyme, the mixture was incubated for 20 min. After removing the protease, the mixture was allowed to return to room temperature, and the pH was adjusted to the optimal reaction pH of each protease using 1 mol / L or NaOH. The enzyme addition amount was 16000 U / g. The reaction conditions for different proteases are shown in Table 1.

[0045] Table 1 Reaction conditions for different proteases

[0046] After the protease hydrolysis reaction was completed, the enzyme activity was eliminated, and the hydrolyzate was cooled to room temperature. It was centrifuged at 5000 rpm for 30 min, and the supernatant was passed through a 0.45 μm filter membrane. After filtration through a 30 kDa ultrafiltration tube, it was stored at -20℃ for later use. The DPPH and ABTS free radical scavenging abilities of the enzyme hydrolyzates of different proteases were determined to select the best enzyme for hydrolysis.

[0047] The results of the determination are shown in Table 2. Figure 1 In the DPPH and ABTS free radical scavenging tests, the free radical scavenging abilities of the five enzyme hydrolyzates were as follows: alkaline protease (87.64%; 73.48%), papain (52.73%; 35.41%), bromelain (27.53%; 28.37%), pepsin (37.74%; 50.61%), and trypsin (22.73%; 32.12%). In summary, the free radical scavenging ability of the enzyme hydrolyzate prepared using alkaline protease was much higher than that of the enzyme hydrolyzates prepared using other proteases. Therefore, alkaline protease was selected as the best enzyme for the preparation of alligator skin collagen peptide.

[0048] 3. Identification and analysis of alligator skin collagen peptide The appropriate crocodile skin collagen protein powder was weighed, and ultrapure water was added at a solid-liquid ratio of 50:1 (mg / mL). The mixture was stirred in a water bath at an appropriate temperature until it was fully dissolved. After the enzyme reached the optimal temperature for hydrolysis, it was incubated for 20 min. The alkaline protease was removed and allowed to return to room temperature. The pH was adjusted to the optimal value for the protease using 1 mol / L or NaOH. The enzyme was added at a concentration of 16000 U / g, and the hydrolysis was performed for 4 h. After inactivation at 95°C for 10 min, the mixture was centrifuged, and then purified using a 3 kDa ultrafiltration tube, a desalting column, and a Sephadex G-50 column to obtain crocodile skin collagen peptides.

[0049] The chemical composition of the crocodile skin collagen peptides was analyzed using ultra-high performance liquid chromatography-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS). The data were processed using Xcalibur 4.1 software (Thermo Fisher Scientific), and the accurate molecular weight values of the detected compounds were matched with the theoretical values. The compounds were analyzed by searching the mz-Cloud database. The mass deviation of the predicted compounds was controlled within ±5 PPM.

[0050] After mass spectrometry identification, the crocodile skin collagen peptides included the following peptide segments: ARLLLAPAK, CCKAQEPEACFK, ELLENEFHQILK, FKNGMLHGQK, KQPKPLK, KSFDVVKHK, LESDVSTQMEYCR, LVTDLTK, NEEQSGDGSR, PHHSPLVLKR, QQFLINLVKQK, SLLPVTK, TILGEQFPKLAK, VGGVGVPAAPSITAVTVNK, VSDALSGDR, WKIAFDKGR, YETELALR. The specific peptide segment information is shown in Table 2, and the molecular weight of the peptide segments is between 700-1800 Da.

[0051] Table 2 Analysis of crocodile skin collagen peptide segments

[0052] The total ion chromatogram of crocodile skin collagen peptides in positive and negative ion modes is shown in Figure 2As shown, the purity of the extracted alligator skin collagen peptide was determined to be 99%. Through preliminary processing by Thermo Xcalibur 4.1 software, through comparison of the theoretical and actual values of the accurate molecular weight, combined with mz-Cloud database retrieval, a total of 20 compounds were identified in the alligator skin collagen peptide, as shown in Table 3. The main components include small molecule peptides, amino acids, and nitrogen-containing metabolites, of which the top five components are: glycyl proline (19.84%): a common dipeptide in collagen degradation; DL-arginine (17.25%): an amino acid with vasodilating and skin repair functions; choline (8.43%): a key molecule for lipid metabolism and cell membrane integrity; glycyl-L-leucine (8.28%): another active dipeptide from collagen; diethyl phosphate (9.64%): possibly derived from phosphate metabolism or exogenous processes. In addition, prolyl leucine, alpha-aspartyl phenylalanine, and cyclo(Leu-Pro) were also detected. These components are typical characteristics of collagen hydrolysates, indicating that the alligator skin collagen peptide is rich in short-chain peptides with potential skin repair and antioxidant functions.

[0053] Table 3 Chemical composition of alligator skin collagen peptide based on ultra-high performance liquid chromatography-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-MS) analysis

[0054] Example 2 A composition containing alligator skin collagen peptide The composition provided in this example contains, by weight, the following components: 5 parts of alligator skin collagen peptide prepared in Example 1, 5 parts of mandelic acid, 10 parts of lactobionic acid, 2 parts of asiaticoside, and 5 parts of nicotinamide.

[0055] The preparation method of the composition is as follows: (1) alligator skin collagen peptide, mandelic acid, lactobionic acid, asiaticoside, and nicotinamide are weighed according to the weight ratio; (2) the above components are mixed with a solvent at room temperature and stirred at 200-300 r / min for 20-30 minutes until completely homogenized to obtain the composition.

[0056] Example 3 A composition containing alligator skin collagen peptide The composition provided in this example contains, by weight, the following components: 8 parts of alligator skin collagen peptide prepared in Example 1, 3 parts of mandelic acid, 12 parts of lactobionic acid, 3 parts of asiaticoside, and 3 parts of nicotinamide. The preparation method of the composition is the same as that of Example 2.

[0057] Example 4 A composition containing alligator skin collagen peptide The composition provided in this embodiment contains, by weight, the following components: 1 part of crocodile skin collagen peptide prepared in Example 1, 8 parts of mandelic acid, 8 parts of lactobionic acid, 1 part of asiaticoside, and 8 parts of nicotinamide. The preparation method of this composition is the same as in Example 2.

[0058] Comparative Example 1 The composition provided in this comparative example contains, by weight, the following components: 0 parts of crocodile skin collagen peptide prepared in Example 1, 5 parts of mandelic acid, 10 parts of lactobionic acid, 2 parts of asiaticoside, and 5 parts of nicotinamide. The preparation method of this composition is the same as in Example 2.

[0059] Comparative Example 2 The composition provided in this comparative example contains, by weight, the following components: 5 parts of crocodile skin collagen peptide prepared in Example 1, 0 parts of mandelic acid, 10 parts of lactobionic acid, 2 parts of asiaticoside, and 5 parts of nicotinamide. The preparation method of this composition is the same as in Example 2.

[0060] Comparative Example 3 The composition provided in this comparative example contains, by weight, the following components: 5 parts of crocodile skin collagen peptide prepared in Example 1, 5 parts of mandelic acid, 0 parts of lactobionic acid, 0 parts of asiaticoside, and 5 parts of nicotinamide. The preparation method of this composition is the same as in Example 2.

[0061] Comparative Example 4 This comparative example was set to contain only the crocodile skin collagen peptide prepared in Example 1.

[0062] Test Example 1: Antibacterial Activity Test 1. Sample to be tested A negative control group (without drug or composition) and a positive control group (with a known effective antibacterial drug) were set up to assess the accuracy and reliability of the experimental results. Multiple concentration gradients of drug or composition solutions were prepared to evaluate the dose-dependent antibacterial effect. The differences between the different compositions in each group are shown in Table 4 below.

[0063] Table 4. Formulation differences between different examples and comparative compositions

[0064] 2. Determination of minimum inhibitory concentration (MIC) The antibacterial effects of different compositions were determined. Compositions of the same concentration and dosage were mixed in liquid agar medium, and their optical density (OD) values ​​were measured using a microplate reader to observe their growth at different concentrations. The microorganism tested was *Propionibacterium acnes*. Propionibacterium acnes Staphylococcus aureus ( Staphylococcus aureus Malassezia ( MalasseziaS. epidermidis, S. aureus, Malassezia respectively use the commercially available products 0.1% Pianolin, phenoxyethanol, 0.1% ZPT as positive control.

[0065] The calculation formula of the inhibition rate is: inhibition rate = experimental group - blank control / negative control - blank control.

[0066] The determination results are shown in Table 5, which shows that the bacteriostatic effect of the composition of Examples 2-4 is significantly better than that of the positive control drugs and Comparative Examples 1-4, wherein the bacteriostatic effect of alligator skin collagen peptide alone in Comparative Example 4 is poor, and the bacteriostatic effect of the composition without alligator skin collagen peptide in Comparative Example 1 is also poor, and the simple superposition of the two cannot achieve the effect of the composition of Examples 2-4, which shows that the compositions of Examples have a certain synergistic effect.

[0067] Table 5 Bacteriostatic activity results

[0068] Test Example 2 Anti-inflammatory activity test 1. Cell inflammatory factor release test With a cell density of 20w cell / mL, 2 mL per well, inoculate Hacat keratinocytes, then add only medium solution in the blank control group, and add LPS in the model group and the test group to induce keratinocytes to release inflammatory factors. The test group is added with culture medium solution containing the same amount of concentration of the test group (the composition of Table 4), and the concentration of the test group is 1 / 16 of the original solution. According to the operation instruction of the ELISA detection kit, the concentration of inflammatory factors (such as IL-1β, IL-6, IL-8) in each well supernatant is detected.

[0069] The determination results are shown in Table 6, which shows that the level of inflammatory factors in the model group is significantly increased, and the level of inflammatory factors in the test group with the addition of the composition of Examples 2-4 under the induction of LPS is significantly reduced, which is significantly better than that of Comparative Examples 1-4. Comparative Example 4 only uses alligator skin collagen peptide, and the anti-inflammatory effect is poor and comparable to the model group. In the examples, the anti-inflammatory function is synergistically enhanced after compounding with other components.

[0070] Table 6 Inflammatory factor content determination results

[0071] 2. Cell toxicity detection (MTT method) The inoculated Hacat cells were placed in a 96-well plate with a density of 8x10 3Cells were divided into a control group and five treatment groups. The control group was cultured in DMEM. After 24 h in a 96-well plate, the culture medium was discarded. The prepared compositions of the examples and comparative examples were diluted halfway with DMEM to five concentration gradients and added to the six treatment groups. The control group was added with DMEM. The plates were then incubated for 24 h. Subsequently, the cell supernatant was removed, and the cells were washed 1-2 times with PBS. 100 μL of 0.5 mg / mL MTT in DMEM solution was added to each well in the dark. After incubating the cell culture plate at 37°C for 4 h, the MTT solution was removed. The cell supernatant was removed, and 150 μL of DMSO was added to each well. After shaking evenly, the absorbance of each well was estimated at 570 nm using a microplate analyzer.

[0072] The test results are shown in Table 7. The results show that the compositions of Examples 2 and 3, when diluted to 1 / 16, were essentially non-toxic to cells, while the compositions of Comparative Examples 1-4, even when diluted to 1 / 32, still retained slight cytotoxicity. This demonstrates that the compositions formulated according to the examples can reduce the irritation of the raw materials and decrease cytotoxicity, reflecting the safety and reliability of the formulation system.

[0073] Table 7 Results of cell viability assay

[0074] Test Example 3: In vitro skin penetration and absorption test 1. Fluorescent labeling A solution of the composition at a certain concentration was prepared, mixed with a Cy5 solution, and reacted in the dark. Unreacted free Cy5 dye was removed by ultrafiltration and dialysis in the dark. The Cy5-labeled sample was then lyophilized for later use. Subsequently, a solution of the Cy5-labeled composition at a certain concentration was prepared using PBS.

[0075] 2. In vitro transdermal absorption test (1) Selection of diffusion pool, ex vivo skin tissue, and receiving pool: A static (Franz) diffusion cell was chosen as the diffusion device. Considering the similarity in physical and chemical structure, the requirements for wound preparation, and the higher permeability of pig skin to hydrophilic molecules compared to human skin, pig skin was selected for the ex vivo skin tissue experiment. Ex vivo pig skin tissue was selected from the back or abdomen of 1-2 month old Bama pigs, with a thickness of less than 1 mm. Fresh or ex vivo pig skin tissue stored at -20°C or lower could be used for the experiment. The receiving cell solution was phosphate-buffered saline (PBS) with a pH of 6.8–7.4, and the recommended temperature was 32°C ± 1°C.

[0076] (2) Preparation of ex vivo skin: Thawed at room temperature naturally before the test, washed with normal saline before use, and the transdermal water loss rate of the skin was detected by a transdermal water loss instrument (transdermal water loss rate <15 g / m 2 ·h}.

[0077] Preparation of wound by skin rolling needle puncture method: take the in vitro pig back / abdominal skin meeting the test requirements, use water-absorbing paper to absorb the water on the front and back of the pig skin, place the horny layer upward on the softwood board covered with paraffin film and stretch (in order to keep the force applied by the microneedle roller approximately constant, place the softwood board with skin on it on a flat surface kitchen scale. Apply the skin rolling needle to all surfaces of the skin, roll once, and keep the rolling force in the range of 4 N~5 N, i.e. 400 g~500 g range on the kitchen scale. According to the area of the diffusion cell and the number required by the test, cut the prepared wound skin into appropriate size for the test).

[0078] Place the matched magnetic sub in the receiving pool of the diffusion cell, and fix the in vitro skin tissue between the supply pool and the receiving pool, and fill the receiving liquid in the receiving pool. Remove the air, and make the bottom surface of the in vitro skin tissue in close contact with the receiving liquid. The stirring rate is selected to achieve sufficient mixing of the sample in the receiving pool without vortex on the surface.

[0079] 3. Experimental grouping In order to remove the error of collagen in pig skin itself, blank control group, positive control group, 0.5 h group, 1 h group, 3 h group, 6 h group were set. Among them, the blank control group: use purified water instead of composition solution sample, prepare blank control according to the formulation and process of the test substance. The blank control group is set at least 2 parallel samples, and the sample group is set at least 4 parallel samples, and the test results are at least 3 groups of effective data. Sample group: take sample in the receiving pool within the corresponding time.

[0080] 4. Transdermal result determination Qualitative detection (tissue section): at least 1 sample parallel sample is applied for qualitative detection. After washing the top part of the test substance, a tissue block of about 10 mm x 10 mm size is taken from the in vitro skin tissue, and appropriate sectioning or grinding technique is used for frozen sectioning. Pay attention to avoid light and reduce fluorescence quenching during operation, and DAPI staining mounting medium can be used for mounting, which is used for positioning the nucleus when fluorescence imaging. In the frozen section sample, in vitro pig skin tissue which has not been prepared for wound preparation should be added as a control for comparison and explanation of wound preparation.

[0081] Quantitative detection: two blank control parallel samples and three sample group parallel samples of the test are applied to quantitative detection. The fluorescence value of the epidermis, dermis and subcutaneous part can be measured by an enzyme label instrument or a fluorescence spectrophotometer. The standard curve of the recombinant collagen concentration-fluorescence intensity is prepared by using the fluorescence-labeled recombinant collagen, and the content of the tested substance in the epidermis, dermis and subcutaneous part of the in-vitro transdermal absorption test is converted.

[0082] Quantitative detection is used to calculate the recovery rate of the tested substance in each diffusion cell in the in-vitro transdermal absorption test. If the recovery rate is within the range of 90% to 110%, it is directly accepted without discussion, which proves that the tested substance is easy to be absorbed through the skin; otherwise, the transdermal efficiency is low. The calculation formula of the recovery rate is as follows: ; In the formula, a0 is the content of the tested substance in the original sample; a1 is the content of the tested substance in the epidermis: the content of the recombinant collagen in the skin tissue surface washing liquid, which can include the skin cleaning liquid, the supply pool cleaning liquid and the like; a2 is the content of the tested substance in the dermis: the content of the recombinant collagen in the skin tissue, which is extracted by using the same component solution (PBS) as the receiving pool after the skin is cut or ground; a3 is the content of the tested substance in the subcutaneous part: the content of the recombinant collagen in the receiving liquid, which includes the receiving liquid at different time points, the receiving pool cleaning liquid and the like.

[0083] The determination results are shown in Table 8. The transdermal effect of the composition formula of Examples 2-4 is significantly better than that of the positive control group, and the recovery rate after 3h is greater than 90%, which indicates that most of the compositions can effectively penetrate the skin and be easily absorbed. The recovery rate of Comparative Examples 2-4 is low, which proves the key and indispensability of each component and the specific ratio in the examples. Since Comparative Example 1 does not contain the labeled collagen, the measured value is 0.

[0084] Table 8 Recovery rate of the tested substance in each diffusion cell in the in-vitro transdermal absorption test

[0085] Test Example 4 Skin repair test Animal modeling experiment of P. acnes was performed: 50 SPF male BALB / c mice were randomly divided into groups (blank control group, model group, test examples 1-7 groups, positive control group (compound dexamethasone acetate cream)) after being depilated, 5 mice in each group were injected with 50 μL of P. acnes suspension intradermally in the ear tissue, and 24 h later, 50 μL of P. acnes suspension was injected again to establish the model.

[0086] The blank control group: no modeling treatment, and no drug application; the model group: modeling but no composition application; the test groups 1-7: 0.1 g of each composition preparation was applied once a day for 6 consecutive days; the positive control group (compound dexamethasone acetate cream): 0.1 g of isotretinoin erythromycin gel was applied once a day for 6 consecutive days. The acne symptoms of the ears of the mice in each group were observed, and the scores were recorded. The specific grouping and treatment methods are shown in Table 9.

[0087] Table 9 Different experimental treatment methods

[0088] The skin inflammation and swelling of the auricle tissue of the mice in each group were observed, and the thickness of the auricle tissue before and after modeling and during the administration period was measured. The auricle swelling degree was calculated according to the following formula, and the auricle swelling inhibition rate was calculated.

[0089] Auricle swelling degree = auricle thickness after the last administration - auricle thickness before administration.

[0090] Auricle swelling inhibition rate (%) = (W 模型 -W 给药 ) / (W 模型 -W 空白 ) x 100%; The auricle swelling degree and inhibition rate of the mice in each group are shown in Table 10 below. After the administration ended, compared with the blank group, the auricle swelling degree of the mice in the model group and the comparative example group was significantly increased, the auricle swelling degree of the mice in the example groups 2-4 was improved to different degrees, and the auricle swelling inhibition rate was significantly higher than that of the positive control group and the comparative example group, indicating that the composition of the example group had good anti-acne efficacy.

[0091] Table 10 Auricle swelling degree and inhibition rate of mice in each group

[0092] The auricle lesion changes of the mice in each group were observed, and HE staining test was performed on the auricle tissue of the mice in the blank group, the model group, the example group 2, and the comparative example group 4. The results are shown in Figure 3As shown, the blank group mice auricle each layer structure is complete and clear, the dermis layer hair follicle and sebaceous gland is rich, no pathological change is found. In sharp contrast, the model group auricle epidermis is abnormal thickening and covered with scab, the dermis and subcutaneous tissue appears necrosis, fibrosis, and accompanied by a large number of inflammatory cell infiltration and tissue edema, structure is seriously disorder. Compared with the model group, the positive group, example 2 group mice auricle tissue pathological injury is reduced, the skin epidermis, dermis layer structure is discernible, the epidermis layer thickening degree is reduced, the dermis and subcutaneous tissue inflammatory cell infiltration is reduced, the relevant HE staining results are as follows Figure 4 As shown, the composition provided by the application has good skin repair effect and can effectively prevent scar formation.

[0093] The above examples are the preferred embodiments of the application, but the embodiments of the application are not limited to the above examples, any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the application should be equivalent replacement methods, and are included in the protection scope of the application.

Claims

1. A crocodile skin collagen peptide, characterized in that, Contains the following peptides: ARLLLAPAK, CCKAQEPEACFK, ELLENEFHQILK, FKNGMLHGQK, KQPKPLK, KSFDVVKHK, LESDVSTQMEYCR, LVTDLTK, NEEQS GDGSR,PHHSPLVLKR,QQFLINLVKQK,SLLPVTK,TILGEQFPKLAK,VGGVGVPAAPSITAVTVNK,VSDALSGDR,WKIAFDKGR,YETELALR.

2. The method for preparing crocodile skin collagen peptides according to claim 1, characterized in that, Includes the following steps: S1. Preparation of crocodile skin collagen: The crocodile skin is cleaned, crushed, degreased and purified, dialyzed to neutral, and freeze-dried to obtain crocodile skin collagen powder for later use. S2. Take an appropriate amount of crocodile skin collagen powder, dissolve it, and then enzymatically hydrolyze it with protease. After enzymatic hydrolysis, inactivate and centrifuge it, then perform ultrafiltration and desalting treatment, and finally purify it to obtain crocodile skin collagen peptides.

3. The preparation method according to claim 2, characterized in that, The enzymatic hydrolysis of proteases uses one or more of the following: alkaline protease, neutral protease, bromelain, pepsin, and trypsin.

4. The preparation method according to claim 2, characterized in that, The enzymatic hydrolysis conditions are: temperature 40~55℃, enzyme addition amount 15000~20000 U / g, and enzymatic hydrolysis for 2~4 h.

5. A composition containing crocodile skin collagen peptides, characterized in that, The composition contains the following components in parts by weight: 0.1-10 parts of the crocodile skin collagen peptide of claim 1, 0.5-15 parts of mandelic acid, 1-20 parts of lactobionic acid, 0.1-5 parts of asiaticoside, and 2-10 parts of nicotinamide.

6. The composition according to claim 5, characterized in that, The composition contains the following components in parts by weight: 1-10 parts of the crocodile skin collagen peptide of claim 1, 1-10 parts of mandelic acid, 5-15 parts of lactobionic acid, 1-5 parts of asiaticoside, and 2-10 parts of nicotinamide.

7. A method for preparing the composition according to claim 5 or 6, characterized in that, Weigh out crocodile skin collagen peptides, mandelic acid, lactobionic acid, asiaticoside and nicotinamide in proportion; mix with solvent at room temperature, and stir at 200-300 r / min for 20-30 minutes until completely homogenized to obtain the composition.

8. The use of the composition of claim 5 or 6 in the preparation of antibacterial, anti-inflammatory, and skin-repairing products.

9. Use of the composition of claim 5 or 6 in the preparation of a medicament for treating acne, folliculitis, seborrhea and acne scars.

10. A product characterized in that, Contains the crocodile skin collagen peptide of claim 1 or the composition of claim 5.

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