A collagen derivative, its preparation method and application

Abstract

This invention relates to a collagen derivative, its preparation method, and its applications. The collagen derivative is a condensation product of compound A and collagen, wherein compound A is an active ingredient containing a carboxyl group. The collagen derivative provided by this invention exhibits excellent penetration-enhancing effects and has broad application prospects in the skincare industry.

Description

Technical Field

[0001] This invention relates to a collagen derivative, its preparation method, and its application. Background Technology

[0002] The skin, the largest organ in the human body, has one of its primary functions: protecting the body from harmful external substances. However, the skin also contains hair follicles, sweat glands, and other organs, serving as an absorption pathway for drugs and cosmetic products. Through application, these substances can be absorbed transdermally, enhancing their efficacy. This is especially important for drugs easily degraded by gastric juices or the intestines, or for anti-aging products that struggle to reach the dermis. To achieve transdermal absorption, various methods have emerged, including physical methods such as ultrasound infusion and microneedling, and chemical methods using azone, ethanol, and borneol. However, these methods have limitations. Physical methods require professional guidance, while chemical methods can thin the stratum corneum, potentially leading to sensitive skin. Therefore, finding a convenient method that does not alter skin structure is crucial for the development of transdermal drugs and skincare products. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a collagen derivative, its preparation method, and its application.

[0004] In a first aspect, the present invention provides a collagen derivative, which is a condensation product of compound A and collagen, wherein compound A is an active ingredient having a carboxyl group.

[0005] According to an embodiment of the present invention, compound A is selected from retinoic acid or its derivatives.

[0006] According to an embodiment of the present invention, compound A is retinoic acid and / or isotretinoin.

[0007] According to an embodiment of the present invention, the average molecular weight of the collagen derivative is 200-1000 kDa.

[0008] According to embodiments of the present invention, the average molecular weight of the collagen is 500 Da to 1,000,000 Da, for example, 800 Da, 1000 Da, 1200 Da, 1400 Da, 1600 Da, 1800 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4500 Da, 5500 Da, 7500 Da, 100,000 Da, 400,000 Da, or 700,000 Da.

[0009] According to an embodiment of the present invention, the average molecular weight of the collagen is 500 Da to 300,000 Da.

[0010] According to an embodiment of the present invention, the average molecular weight of the collagen is 500 Da to 5000 Da.

[0011] According to an embodiment of the present invention, the average molecular weight of the collagen is 500 to 1500 Da.

[0012] In a second aspect, the present invention provides a method for preparing a collagen derivative, comprising condensing collagen with compound A to generate a collagen derivative.

[0013] According to an embodiment of the present invention, compound A is retinoic acid or a derivative thereof.

[0014] According to an embodiment of the present invention, compound A is retinoic acid and / or isotretinoin.

[0015] According to an embodiment of the present invention, the molar ratio of collagen to compound A is 1:(0.01 to 1); for example, 1:0.01, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, or 1:1.

[0016] According to embodiments of the present invention, the average molecular weight of the collagen is 500 Da to 1,000,000 Da, for example, 800 Da, 1000 Da, 1200 Da, 1400 Da, 1600 Da, 1800 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4500 Da, 5500 Da, 7500 Da, 100,000 Da, 400,000 Da, or 700,000 Da.

[0017] According to an embodiment of the present invention, the average molecular weight of the collagen is 500 Da to 300,000 Da.

[0018] According to an embodiment of the present invention, the average molecular weight of the collagen is 500 Da to 5000 Da.

[0019] According to an embodiment of the present invention, the average molecular weight of the collagen is 500 to 1500 Da.

[0020] According to an embodiment of the present invention, the condensation reaction uses EDC and NHS as condensation reagents.

[0021] According to an embodiment of the present invention, the molar ratio of compound A to EDC is 1:(0.5 to 1.5); for example, 1:0.5, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5.

[0022] According to an embodiment of the present invention, the molar ratio of compound A to NHS is 1:(0.5 to 1.5); for example, 1:0.5, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5.

[0023] According to an embodiment of the present invention, the condensation reaction takes 0.1 to 24 hours.

[0024] According to an embodiment of the present invention, the temperature of the condensation reaction is -5 to 10°C, for example -5°C, -2°C, 0°C, 1°C, 2°C, 3°C, 4°C, 6°C, 8°C, or 10°C.

[0025] According to an embodiment of the present invention, the temperature of the condensation reaction is 0 to 4°C.

[0026] According to an embodiment of the present invention, compound A is dissolved in a mixed solution of PBS and tetrahydrofuran before the condensation reaction.

[0027] According to an embodiment of the present invention, the pH of the PBS solution is 2 to 6, for example, 2, 3, 4, 5, or 6.

[0028] According to an embodiment of the present invention, the concentration of the PBS solution is 0.05 to 1 M, for example, 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, or 1 M.

[0029] According to an embodiment of the present invention, the volume ratio of the PBS solution to tetrahydrofuran is 1:(0.5-2), for example, 1:0.5, 1:1, 1:1.3, 1:1.5, 1:1.7, or 1:2.

[0030] According to an embodiment of the present invention, the preparation method includes: dissolving compound A in a mixed solution of PBS (0.05M-1M) and tetrahydrofuran (1:0.5-2) at pH 2-6, adding EDC and NHS, activating at 0-4°C for 0.1-4 h, then adding collagen, and controlling the temperature for 0-24 h of reaction; after the reaction is completed, dialysis (MW-3000Da) and freeze-drying to obtain a collagen derivative.

[0031] In a third aspect, the present invention provides the application of the collagen derivatives described in the first aspect of the present invention or the collagen derivatives prepared by the preparation method described in the second aspect of the present invention in the field of skin care products.

[0032] In a fourth aspect, the present invention provides a skin care product comprising an active ingredient and a collagen derivative prepared by the preparation method described in the first aspect of the present invention or the preparation method described in the second aspect of the present invention.

[0033] According to embodiments of the present invention, the active ingredient is a skin-care active ingredient, such as a whitening or acne-removing ingredient, including but not limited to: tranexamic acid, niacin, niacinamide, salicylic acid, etc.

[0034] According to an embodiment of the present invention, the skin care product may be water, lotion, cream, mask, etc.

[0035] According to an embodiment of the present invention, the mass ratio of the collagen derivative to the active ingredient is 1:(0.01-10), for example, 1:0.01, 1:0.1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0036] Collagen, as a biological macromolecule, is widely present in the connective tissues of animals and is also widely distributed in various tissues and organs of mammals, thus exhibiting excellent biocompatibility and biosafety. Retinoic acid, as a metabolite of vitamin A, mainly affects bone growth and promotes epithelial cell proliferation, differentiation, and keratinization. Furthermore, nanoparticles can penetrate the skin through the gaps in the stratum corneum. Based on these considerations, this invention designs and synthesizes a collagen derivative that, utilizing the amphiphilic nature of its molecular structure, can self-assemble into nanoparticles in aqueous solution. Furthermore, the unique properties of these nanoparticles can promote the transdermal absorption of its active ingredients.

[0037] The collagen derivative of this invention increases the interaction between the collagen and lipids in the stratum corneum of the skin, promoting a reduction in stratum corneum resistance and improving its transdermal efficiency. By regulating the ratio of collagen to compound A, a structurally tunable collagen derivative is obtained. The collagen derivative of this invention can effectively promote the transdermal absorption of active ingredients in skincare products and has broad application prospects in the skincare field. Attached Figure Description

[0038] Figure 1 The nuclear magnetic resonance spectrum of the collagen derivative of Example 1 of the present invention;

[0039] Figure 2 The infrared spectrum of the collagen derivative of Example 1 of the present invention;

[0040] Figure 3 This is a fluorescence imaging image of the transdermal experiment in Application Example 1 of the present invention;

[0041] Figure 4 for Figure 3 A statistical graph of the fluorescence imaging results. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0043] The collagen used in these embodiments of the invention is hydrolyzed collagen with a molecular weight distribution of 500-1500 Da or 2000-3000 Da. The collagen used in these embodiments of the invention can be obtained through purchase.

[0044] Example 1

[0045] Dissolve 3g of retinoic acid in 30mL of a mixed solution of pH=5 (0.1M) PBS and 30mL of tetrahydrofuran. Add 2.2g of EDC and 1.6g of NHS in an ice bath (0℃) and react for 2 hours. Then add 5g of collagen (500-1500Da) and continue the reaction at room temperature for 12 hours. After dialysis (MW=500Da), freeze-dry to obtain 4.5g of product.

[0046] Example 2

[0047] Dissolve 6g of retinoic acid in 50mL of a mixed solution of pH=4 (0.1M) PBS and 50mL of tetrahydrofuran. Add 6g of EDC and 4.5g of NHS in an ice bath (0℃) and react for 1 hour. Then add 15g of collagen (2000-3000Da) and continue the reaction at room temperature for 12 hours. After dialyzing (MW=500Da), freeze-dry to obtain 6.8g of product.

[0048] Example 3

[0049] Dissolve 3g of isotretinoin in 50mL of a mixed solution of pH=4 (0.1M) PBS and 50mL of tetrahydrofuran. Add 3g of EDC and 2.3g of NHS in an ice bath (0℃) and react for 1 hour. Then add 5g of collagen (500-1500Da) and continue the reaction at room temperature for 12 hours. After dialyzing (MW=500Da), freeze-dry to obtain 4.8g of product.

[0050] Figure 1 This is the proton spectrum of Example 1. Figure 2 This is the infrared spectrum of Example 1, from Figure 1 and Figure 2 It can be seen that the collagen derivative of the present invention contains amide bonds.

[0051] Application Example 1: Transdermal Efficacy Test

[0052] All solvents and reagents were of chromatographic grade, and standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd., as detailed in Tables 1 and 2.

[0053] Table 1

[0054] reagents Manufacturer Trifluoroacetic acid (mass spectrometry grade) Sigma Aldrich (Shanghai) Trading Co., Ltd. Acetonitrile (mass spectrometry grade) Sigma Aldrich (Shanghai) Trading Co., Ltd. Ultrapure water After purification using the Millipore pure water system, the resistivity reaches 18.2 MΩ·cm.

[0055] Table 2

[0056]

[0057] The sample processing steps are as follows: Add the sample to ultrapure water according to the formula in Table 3, centrifuge at 12000 r / min for 10 min; take the supernatant into the inner liner tube and detect it by HPLC.

[0058] The HPLC detection method is as follows:

[0059] The instrument used in this experiment was a Waters 2695 high-performance liquid chromatograph equipped with a Waters 2424 evaporative light detector. The chromatographic column was a Diamonsil C18(2) (150*4.6mm, 5μm), the mobile phase was acetonitrile:0.3% TFA water / 7:93, the drift tube temperature was 85℃, the nebulizer temperature was 30℃, the nitrogen flow rate was 25psi, the gain was 100, the mobile phase flow rate was 1mL / min, the column temperature was 35℃, and the injection volume was 10μL.

[0060] Table 3

[0061]

[0062]

[0063] The results show that using the collagen derivative of the present invention as a penetration enhancer mixed with the active ingredient tranexamic acid can significantly improve the penetration rate of the active ingredient; moreover, under the same conditions, the collagen derivative of the present invention has a superior penetration-enhancing effect compared with other reference materials.

[0064] Application Example 2

[0065] 1. Sample preparation

[0066] The collagen derivative in Example 1 was labeled with the fluorescent probe rhodamine to obtain labeled collagen derivative (Rho-Col-VA), and a 5 mL aqueous solution A containing 0.1 wt% labeled collagen derivative was prepared.

[0067] Collagen (500-1500 Da) was labeled with the fluorescent probe fluorescein isothiocyanate (FITC) to obtain labeled collagen (FITC-Col). An aqueous solution containing 0.1 wt% labeled collagen was prepared as control group B.

[0068] 2. Transdermal test

[0069] Twenty C57BL / 6 mice were randomly divided into two groups of ten each: a collagen derivative group and a collagen group. Two 5*5cm patches of hair were removed from each mouse using an electric razor, exposing the skin. Then, 0.5 mL of the prepared collagen derivative group A and collagen group B were applied to the two shaved skin patches, respectively. Eight hours later, the mice were anesthetized and euthanized. Skin samples from both groups were collected, and paraffin sections were prepared for fluorescence imaging. Figure 3 As shown.

[0070] 3. Quantitative PCR methods

[0071] Data were analyzed using SPSS 13.0 statistical software. The interpretation results of the direct immunofluorescence staining method for frozen sections were compared using the chi-square test, with P < 0.05 considered statistically significant. Results are as follows: Figure 4 As shown. It should be noted that, Figure 4 In the text, collagen FITC-Col represents... Figure 3 Absorption of FITC-Col; Collagen derivative (Example 1) FITC-Col-VA representation Figure 3 The amount of FITC-Col-VA absorbed.

[0072] 4. Results

[0073] from Figure 4 It can be concluded that the collagen derivative of the present invention can significantly promote transdermal absorption by the skin.

[0074] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A collagen derivative, which is a condensation product of compound A and collagen, wherein the compound A is an efficacy ingredient having a carboxyl group. The compound A is tretinoin. The collagen has an average molecular weight of 500 Da to 1500 Da or 2000 Da to 3000 Da. 2.A method for preparing a collagen derivative, comprising condensing collagen with compound A to generate the collagen derivative, wherein the compound A is tretinoin, and the collagen has an average molecular weight of 500 Da to 1500 Da or 2000 Da to 3000 Da.

3. The production method according to claim 2, characterized by, The molar ratio of the collagen to the compound A is 1: (0.01 to 1).

4. The production method according to claim 2, characterized by, The condensation reaction uses EDC and NHS as condensation reagents; the molar ratio of the compound A to the EDC is 1: (0.5 to 1.5) ; and the molar ratio of the compound A to the NHS is 1: (0.5 to 1.5).

5. The preparation method according to claim 2, characterized in that, The condensation reaction has a time of 0.1 to 24 hours, and / or a temperature of -5 to 10 ℃.

6. The preparation method according to claim 2, characterized in that, The condensation reaction has a time of 0.1 to 4 hours.

7. The preparation method according to claim 2, characterized in that, The condensation reaction has a temperature of 0 to 4 ℃.

8. The preparation method according to claim 2, characterized in that, The compound A is dissolved in a mixed solution of PBS solution and tetrahydrofuran before the condensation reaction, wherein the PBS solution has a concentration of 0.05 to 1 M, and the volume ratio of the PBS solution to tetrahydrofuran is 1: (0.5 to 2).

9. The production method according to claim 8, characterized by, The PBS solution has a pH of 2 to 6. 10.Use of the collagen derivative of claim 1 or the collagen derivative prepared by the method of any one of claims 2 to 9 in the preparation of a skin care product. 11.A skin care product comprising an efficacy ingredient and the collagen derivative of claim 1 or the collagen derivative prepared by the method of any one of claims 2 to 9.

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