Preparation method and application of collagen and Ectoin eutectic

Abstract

The invention provides a preparation method and application of a collagen and Ectoine eutectic, collagen and Ectoine are sequentially put into a solvent to be uniformly mixed, and then the mixture is dried at the temperature of 40-50 DEG C to obtain the collagen and Ectoine eutectic. The invention finds that recombinant XVII type collagen and Ectoine can form a new supramolecular eutectic substance in a non-covalent bond binding manner. The supramolecular co-crystal has the effects of improving solubility and compatibility, improving efficacy absorbability, enhancing stability, improving safety and the like. Through the synergistic effect of the Ectoin and the collagen, the skin permeability can be improved, and the skin care effect and the anti-inflammatory effect can be synergistically enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a preparation method of a collagen and ectoine eutectic and application thereof. Background Art

[0002] Collagen is a major structural protein in the extracellular matrix of mammals and is widely present in skin, bones, muscles and other tissues. Compared with animal-derived collagen extracted by traditional methods, recombinant type XVII collagen has better water solubility, stronger processability and a single component. The triple-helix structure of recombinant type XVII collagen has shown excellent effects in promoting cell adhesion, migration, epidermal repair, regeneration of hair follicle stem cells, and improvement of hair density and growth rate. Non-fibrous recombinant type XVII collagen is gradually emerging in the field of skin anti-aging and is a type of active molecule with broad application prospects.

[0003] Ecdoin, also known as tetrahydropyrimidine, is an amino acid derivative that exists in many organisms. It can provide a strong protective barrier for cells under harsh conditions such as high temperature, dryness, and strong radiation, maintain cell stability and vitality, and help organisms survive under extreme conditions. It is a common ingredient in cosmetics and Western medicine. Ecdoin has excellent moisturizing, repairing and protective effects. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides a method for preparing a collagen-ectoine eutectic, which comprises the following steps:

[0006] The collagen and ectoine are sequentially put into a solvent and mixed evenly, and then dried at 40-50° C. to obtain a eutectic of collagen and ectoine.

[0007] As a preferred embodiment of the method for preparing the collagen and ectoine co-crystal of the present invention: the collagen is human recombinant type XVII collagen.

[0008] As a preferred embodiment of the method for preparing the collagen and ectoine eutectic of the present invention, the mass ratio of the collagen to ectoine is 1:3-5.

[0009] As a preferred embodiment of the method for preparing the collagen and ectoine eutectic of the present invention: the solvent includes water, and the collagen and ectoine are sequentially added into the solvent, and the material-liquid ratio is 1:1-20.

[0010] As a preferred embodiment of the method for preparing the collagen and ectoine eutectic of the present invention, the uniform mixing includes stirring at 20 to 30° C. and 200 to 300 rpm for 10 to 60 minutes.

[0011] As a preferred embodiment of the method for preparing the collagen and ectoine eutectic of the present invention, the collagen and ectoine are sequentially added into a solvent, and the solid-liquid ratio is 1:5-10.

[0012] Beneficial effects of the present invention: The present invention finds that recombinant type XVII collagen and ectoine can form a new supramolecular eutectic substance by non-covalent bonding. The supramolecular eutectic has the effects of improving solubility and compatibility, improving efficacy absorption, enhancing stability, and improving safety. The synergistic effect of ectoine and collagen can not only improve skin permeability, but also synergistically enhance skin care effects and anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0014] Figure 1 This is the XRD spectrum of the co-crystal of recombinant type XVII collagen and ectoine.

[0015] Figure 2 This is the infrared spectrum of the co-crystal of recombinant type XVII collagen and ectoine.

[0016] Figure 3 This is a graph showing the penetration of recombinant type XVII collagen and ectoine co-crystal into pig skin at different times.

[0017] Figure 4 This is a graph showing the proliferation rate of RAW264.7 cells.

[0018] Figure 5 This graph shows the effects of the co-crystal and mixture on the LPS-induced inflammatory factor NO.

[0019] Figure 6 This graph shows the effects of co-crystals and mixtures on LPS-induced inflammatory factor TNF-α.

[0020] Figure 7 This graph shows the effects of co-crystals and mixtures on LPS-induced inflammatory factor IL-6. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0022] Embodiment 1:

[0023] Recombinant type XVII collagen powder (CAS: 9064-67-9, humanized recombinant collagen) and ectoine (CAS No.: 96702-03-3) are sequentially added into deionized water, the mass ratio of recombinant type XVII collagen and ectoine is 1:4, and the solid-liquid ratio is 1:10 (g / g). Then, the mixture is stirred at 20°C and 200-300rpm for half an hour. After stirring, it is dried in an oven at 40°C to obtain a solid powder of the eutectic.

[0024] Preparation of physical mixing group samples: recombinant type XVII collagen powder and ectoine were mixed at a mass ratio of 1:4, and ground with a pestle for 10 minutes to obtain control group samples.

[0025] The following experimental tests were performed on the above eutectic solid powder:

[0026] (1) The diffraction patterns of the samples were collected by X-ray diffractometer (XRD), and the specific crystal structure was identified based on the position and intensity of the peaks in the pattern. Different types of crystals have unique lattice parameters and atomic arrangements, which cause X-rays to diffract at specific angles, producing unique diffraction peak patterns. Ectoin and collagen, their mixtures and cocrystals were analyzed by XRD, using a copper target (λ = 0.154 nm), a scanning speed of 5° / min, and a scanning range of 5°-60° (2 θ ).

[0027] (2) The main function of Fourier transform infrared spectrometer (FTIR) is to analyze the macroscopic chemical information of materials. FTIR is used to analyze the chemical information of ectoine and collagen, their mixtures and co-crystals.

[0028] (3) Skin penetration test:

[0029] Using the improved Franz diffusion cell method, the pig skin was fixed between the supply cell and the receiving cell, with the stratum corneum facing the supply cell. The receiving solution was a phosphate buffer solution with a pH of 6.8, and the receiving cell was kept in a constant temperature water bath at (37±0.2)°C. After adding the phosphate buffer solution to the receiving cell, magnetic stirring was performed at a speed of 300r / min to eliminate the bubbles generated in the subcutaneous layer of the pig, and the receiving cell was filled with the receiving solution with a syringe. The eutectic was prepared into a 10mg / mL aqueous solution, 1mL of the eutectic solution was added to the supply cell, and 0.75mL of the receiving solution was taken at 1, 2, 4, and 8h, respectively. At the same time, the receiving cell was supplemented with an equal volume of blank receiving solution at the same temperature. After filtering the removed receiving solution, its content was determined, and the cumulative permeation amount Q at each time point was calculated according to the following formula n (μg / cm 2 ):

[0030]

[0031] C n is the mass concentration of the eutectic in the receiving solution at the nth sampling point, V is the volume of the receiving pool (7 mL), V0 is the sampling volume at each time point, C i is the mass concentration of eutectic in the receiving solution at the i-th sampling point (i≤n-1), and A is the effective diffusion cell area (2.2cm 2 ). The cumulative penetration amount Q n Plot Qt against time t and perform model fitting.

[0032] (4) RAW264.7 cell proliferation rate test:

[0033] RAW264.7 cells in the logarithmic growth phase were digested, centrifuged, and counted using a hemocytometer. 1×10 cells were seeded per well of a 96-well plate. 4 cells and cultured them at 37°C and 5% CO2 for 24 hours. Then discard the supernatant, add different concentrations of co-crystals dissolved in culture medium and physically mixed sample solutions of collagen and ectoine to the sample wells, respectively, with no sample as the blank control group, and set up multiple replicates for each well. After 24 hours of culture, discard the supernatant and wash with PBS, and add 100 μL of freshly prepared MTT solution of 0.5 mg / mL (dissolved in DMEM culture medium) to each well. After incubation for 4 hours, carefully aspirate the supernatant in the well, add 100 μL DMSO to each well, oscillate at 37°C for 5 minutes, select 490nm wavelength to measure the absorbance of each well, and the cell proliferation calculation formula is:

[0034]

[0035] Wherein: A0 represents the absorbance value of the solvent group; A1 represents the absorbance value of the sample group; A2 represents the absorbance value of the blank control group.

[0036] (5) Establishment of inflammation model:

[0037] LPS is a component of the outer wall of Gram-negative bacteria and a typical stimulant that can induce macrophage activation and increase the level of inflammatory factors. Using the inflammatory factor NO as an indicator, an inflammatory model was established to investigate the anti-inflammatory effect of SF. DMEM high-glucose medium was used as the control group, 1 μg mL -1 The model group was composed of LPS as a stimulus, and the experimental group was composed of LPS and co-crystals of different mass concentrations. After 24 hours of culture, the supernatant was aspirated and the contents of NO, TNF-α, and IL-6 were determined according to the procedures of the kit.

[0038] The test results of the co-crystal are as follows:

[0039] (1) XRD spectrum of recombinant type XVII collagen and ectoine co-crystal is shown in Figure 1 The characteristic peaks of ectoine are 11.63°, 12.46°, 15.86°, 18.06°, 19.62°, 20.55°, 23.27°, 25.17°, 25.96°, 26.71°, 27.17°, 28.86° and 35.64°, and there is a broad peak of recombinant type XVII collagen near 21.28°. After the reaction of recombinant type XVII collagen and ectoine, the XRD spectrum changed. The characteristic peaks of the raw material recombinant type XVII collagen and ectoine were weakened or disappeared to varying degrees. It can be seen from the figure that 2 in the XRD spectrum of the co-crystal θ New characteristic peaks appeared at angles of 13.55°, 17.01°, 21.53°, 22.46°, 24.58°, 31.06°, 32.76°, 33.78° and 36.92°, while no new characteristic peaks appeared in the material physically mixed with recombinant type XVII collagen and ectoine, which means that this method can successfully prepare recombinant type XVII collagen and ectoine co-crystals.

[0040] (2) Infrared images of recombinant type XVII collagen and ectoin Figure 2 , the OH of ectoine is at 3183cm -1 The carbonyl C=O shows stretching vibration at 1588cm -1 After the formation of recombinant type XVII collagen and ectoine cocrystal, the OH group of ectoine red-shifted to 3181 -1 , C=O red shifted to 1581cm -1 The above shows that there is a weak hydrogen bond between ectoine and collagen, which provides evidence for the formation of a eutectic supramolecular between ectoine and collagen.

[0041] (3) Cumulative permeation of recombinant type XVII collagen and adenosine co-crystal (Q n ) and time (t) as shown in the figure Figure 3 As shown in Figure 2, the cumulative permeation amount (Q n ) increased, and the cumulative permeation gradually stabilized with the increase of time. The cumulative permeation of the co-crystal at the four sampling times of 1, 2, 4, and 8 hours was higher than that of recombinant type XVII collagen. The cumulative permeation of 8 hours of transdermal permeation increased by 29.4% compared with recombinant type XVII collagen, indicating that the co-crystal has a good permeation-enhancing effect.

[0042] (4) Figure 4The co-crystal has a proliferative effect on RAW264.7 cells. The co-crystal solution was non-toxic to cells in the concentration range of 0.05%-0.5%, and compared with the mixture, the co-crystal showed a significant pro-proliferation effect on RAW264.7 cells at 0.2% and 0.5% (p<0.05), with proliferation rates of 120.65% and 128.15%, respectively, indicating that the co-crystal has a good skin repair effect.

[0043] (5) Figure 6 , Figure 7 The effects of the co-crystal and the mixture on the expression of multiple inflammatory factors secreted by RAW264.7 cells when stimulated, including nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The effects of the co-crystal and the mixture on the expression of three inflammatory factors (NO, TNF-α, IL-6) were detected using a kit. The results are as follows Figure 6-7 As shown in the results, the 0.5% co-crystal reduced the levels of NO, TNF-α and IL-6 in LPS-induced human immortalized keratinocytes by 54.02%, 55.90% and 61.2%, respectively. The co-crystal had a significantly better inhibitory effect on inflammatory factors than the mixture, and this inhibitory effect was dose-dependent, indicating that the co-crystal had good anti-inflammatory function.

[0044] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a collagen-ectoine eutectic, characterized in that: It consists of the following steps: The collagen and ectoine are sequentially put into a solvent and mixed evenly, and then dried at 40-50° C. to obtain a eutectic of collagen and ectoine.

2. The method for preparing the collagen and ectoine eutectic according to claim 1, characterized in that: The collagen is human recombinant XVH type collagen.

3. The method for preparing the collagen and ectoine eutectic according to claim 1 or 2, characterized in that: The mass ratio of the collagen to ectoine is 1:3-5.

4. The method for preparing the collagen and ectoine eutectic according to claim 1 or 2, characterized in that: The solvent includes water, and the collagen and ectoine are sequentially added into the solvent, and the material-liquid ratio is 1:1-20.

5. The method for preparing the collagen and ectoine eutectic according to claim 1 or 2, characterized in that: The uniform mixing includes stirring at 20-30° C. and 200-300 rpm for 10-60 minutes.

6. The method for preparing the collagen and ectoine eutectic according to claim 1 or 2, characterized in that: The collagen and ectoine are sequentially added into the solvent, and the solid-liquid ratio is 1:5-10.

7. The collagen and ectoine eutectic prepared by the method for preparing the collagen and ectoine eutectic according to claim 1.

8. Use of the collagen and ectoine eutectic prepared by the preparation method of collagen and ectoine eutectic according to claim 1 in preparing skin repair products.

9. Use of the collagen and ectoine eutectic prepared by the preparation method of the collagen and ectoine eutectic according to claim 1 in the preparation of anti-inflammatory products.

10. Use of the collagen and ectoine eutectic prepared by the method for preparing the collagen and ectoine eutectic according to claim 1 in the preparation of cosmetics.

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