Collagen derivative as well as preparation method and application thereof

Abstract

The invention relates to a collagen derivative as well as a preparation method and application thereof. The collagen derivative is a condensation product of a compound A and collagen, wherein the compound A is a functional component with carboxyl. The collagen derivative provided by the invention has a good permeation promoting effect and has a wide application prospect in the field of skin care products.

Description

Technical Field

[0001] The invention relates to a collagen derivative and a preparation method and application thereof. Background Art

[0002] The skin is the largest organ of the human body, and one of its main functions is to protect the human body from harmful substances from the outside world. On the other hand, the skin can also be an absorption pathway for drugs or daily chemical products due to the presence of hair follicles, sweat glands and other organs. Through smearing and other methods, the percutaneous absorption of drugs or daily chemical products can be achieved, thereby improving the efficacy of drugs or daily chemical products. This is especially true for some drugs that are easily degraded by gastric juice or intestinal tract, or anti-aging products that are difficult to reach the dermis of the skin. In order to achieve the goal of percutaneous absorption, there are currently a variety of percutaneous absorption methods, such as physical methods, including ultrasound introduction, microneedle injection, etc.; such as chemical methods, such as azone, ethanol, borneol, etc. However, the use of the above methods has its limitations. For example, physical methods require the guidance of professionals, while chemical methods are likely to cause the thinning of the skin stratum corneum, ultimately leading to sensitive skin. Therefore, finding a method that is easy to use and does not cause changes in skin structure is of great significance for the development of percutaneous drugs and skin care products. Summary of the invention

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

[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 effective 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 tretinoin and / or isotretinoin.

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

[0008] According to an embodiment of the present invention, the average molecular weight of the collagen is 500Da to 1 million Da, for example, 800Da, 1000Da, 1200Da, 1400Da, 1600Da, 1800Da, 2000Da, 2500Da, 3000Da, 3500Da, 4500Da, 5500Da, 7500Da, 100000Da, 400000Da, or 700000Da.

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

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

[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 subjecting collagen to a condensation reaction with a compound A to generate a collagen derivative.

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

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

[0015] According to an embodiment of the present invention, the molar ratio of the collagen to the compound A is 1:(0.01-1); 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, 1:1.

[0016] According to an embodiment of the present invention, the average molecular weight of the collagen is 500Da to 1 million Da, for example, 800Da, 1000Da, 1200Da, 1400Da, 1600Da, 1800Da, 2000Da, 2500Da, 3000Da, 3500Da, 4500Da, 5500Da, 7500Da, 100000Da, 400000Da, or 700000Da.

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

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

[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 the compound A to the EDC is 1:(0.5-1.5); for example, 1:0.5, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5.

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

[0023] According to an embodiment of the present invention, the condensation reaction time is 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 0-4°C.

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

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

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

[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 comprises: dissolving compound A in a mixed solution of a PBS solution (0.05M-1M) with a pH of 2-6 and tetrahydrofuran (1:0.5-2), adding EDC and NHS thereto, activating at 0-4°C for 0.1-4h, then adding collagen thereto, controlling the temperature to react for 0-24h; after the reaction is completed and dialyzed (MW-3000Da), a collagen derivative is obtained after freeze-drying.

[0031] In a third aspect, the present invention provides use of the collagen derivative described in the first aspect of the present invention or the collagen derivative 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 effective ingredient and the collagen derivative according to the first aspect of the present invention or the collagen derivative prepared by the preparation method described in the second aspect of the present invention.

[0033] According to an embodiment of the present invention, the functional ingredients are functional ingredients for skin, such as whitening and acne-removing ingredients, including but not limited to: tranexamic acid (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, facial mask, etc.

[0035] According to an embodiment of the present invention, the mass ratio of the collagen derivative to the effective 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, 1:10.

[0036] As a biopolymer, collagen is widely present in the connective tissue of animals and is also widely distributed in various tissues and organs of mammals, so it has excellent biocompatibility and biosafety. As a metabolite of vitamin A, retinoic acid mainly affects bone growth and promotes metabolic effects such as epithelial cell proliferation, differentiation, and keratolysis. In addition, nanoparticles can enter the skin through the gaps in the stratum corneum of the skin. Based on the above considerations, the present invention designs and synthesizes a collagen derivative, which can self-assemble into nanoparticles in an aqueous solution by utilizing the amphiphilic characteristics of the molecular structure itself. The unique properties of its nanoparticles can be used to promote the percutaneous absorption of its functional substances.

[0037] The collagen derivative of the present invention increases the interaction with lipids in the stratum corneum of the skin, promotes the reduction of the resistance of the stratum corneum of the skin, and improves its transdermal efficiency; by adjusting the ratio of collagen and compound A, a collagen derivative with adjustable structure is obtained. The collagen derivative of the present invention can effectively promote the transdermal absorption of the functional ingredients in skin care products, and has broad application prospects in the field of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the NMR spectrum of the collagen derivative of Example 1 of the present invention;

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

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

[0041] Figure 4 for Figure 3 Statistical graph of fluorescence imaging results. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

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

[0044] Example 1

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

[0046] Example 2

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

[0048] Example 3

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

[0050] Figure 1 is the hydrogen spectrum of Example 1, Figure 2 is the infrared spectrum of Example 1, Figure 1 and Figure 2 It can be seen that the collagen derivative of the present invention contains an amide bond.

[0051] Application Example 1: Transdermal effect test:

[0052] All solvents and reagents were of chromatographic grade, and standard products were purchased from Shanghai Yuanye Biotechnology Co., Ltd., as shown 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 Purified by Millipore pure water system, the resistivity reaches 18.2MΩ·cm

[0055] Table 2

[0056]

[0057] The sample processing steps are as follows: add the formula in Table 3 into ultrapure water, centrifuge at 12000r / min for 10min; take the supernatant into the inner liner tube and detect 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 Diamonsil C18 (2) (150*4.6 mm, 5 μm), the mobile phase was acetonitrile: 0.3% TFA water / 7:93, the drift tube temperature was 85°C, the nebulizer temperature was 30°C, the nitrogen flow rate was 25 psi, the gain was 100, the mobile phase flow rate was 1 mL / min, the column temperature was 35°C, and the injection volume was 10 μL.

[0060] Table 3

[0061]

[0062]

[0063] The results show that the use of the collagen derivative of the present invention as a permeation enhancer mixed with the active ingredient tranexamic acid can significantly increase the permeability of the active ingredient; and, under the same conditions, compared with other reference substances, the permeation enhancement effect of the collagen derivative of the present invention is more excellent.

[0064] Application Example 2

[0065] 1. Test sample preparation

[0066] The collagen derivative in Example 1 was labeled with the fluorescent probe rhodamine to obtain a labeled collagen derivative (Rho-Col-VA), and 5 mL of aqueous solution A containing 0.1 wt % of the 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, 10 mice in each group, namely the collagen derivative group and the collagen group. Two 5*5 cm hair patches of each mouse were removed with an electric shaver to expose the skin, and then 0.5 mL of the prepared collagen derivative group A and collagen group B were applied to the two hairless skin patches. After eight hours, the mice were anesthetized and killed, and the skins applied with collagen derivative group A and collagen group B were taken, paraffin sections were prepared routinely, and fluorescence imaging was performed, such as Figure 3 shown.

[0070] 3. Fluorescence quantitative statistical method

[0071] SPSS13.0 statistical software was used to analyze the data. The interpretation results of the direct immunofluorescence staining method of frozen sections were compared using the x2 test, and P < 0.05 was considered statistically significant. Figure 4 It should be noted that Figure 4 In the middle, collagen FITC-Col represents Figure 3 Absorption in FITC-Col; collagen derivative (Example 1) FITC-Col-VA indicates Figure 3 The absorption of FITC-Col-VA in .

[0072] 4. Results

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

[0074] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A collagen derivative, which is a condensation product of compound A and collagen, wherein compound A is an effective ingredient having a carboxyl group.

2. The collagen derivative according to claim 1, characterized in that Compound A is selected from retinoic acid or its derivatives, preferably retinoic acid and / or isotretinoin.

3. The collagen derivative according to claim 1 or 2, characterized in that: The average molecular weight of the collagen derivative is 200 to 1000 KDa; and / or The average molecular weight of the collagen is 500 Da to 1,000,000 Da, preferably 500 Da to 300,000 Da, more preferably 500 Da to 5,000 Da, and even more preferably 500 to 1,500 Da.

4. A method for preparing a collagen derivative, comprising subjecting collagen to a condensation reaction with a compound A to generate a collagen derivative.

5. The preparation method according to claim 4, characterized in that: The compound A is tretinoin or a derivative thereof, preferably, the compound A is tretinoin and / or isotretinoin; and / or, the molar ratio of the collagen to the compound A is 1:(0.01-1); And / or, the average molecular weight of the collagen is 500Da to 1,000,000Da, preferably 500Da to 300,000Da, more preferably 500Da to 5,000Da, and further preferably 500 to 1,500Da.

6. The preparation method according to claim 4 or 5, characterized in that: The condensation reaction uses EDC and NHS as condensation reagents; and / or, the molar ratio of the compound A to the EDC is 1:(0.5-1.5); and / or, the molar ratio of the compound A to the NHS is 1:(0.5-1.5).

7. The preparation method according to any one of claims 4 to 6, characterized in that: The condensation reaction time is 0.1 to 24 hours, preferably 0.1 to 4 hours, and / or the condensation reaction temperature is -5 to 10°C, preferably 0 to 4°C.

8. The preparation method according to any one of claims 4 to 7, characterized in that: Before the condensation reaction, the compound A is first dissolved in a mixed solution of PBS solution and tetrahydrofuran. Preferably, the pH of the PBS solution is 2-6, and / or the concentration of the PBS solution is 0.05-1M, and / or the volume ratio of the PBS solution to tetrahydrofuran is 1:(0.5-2).

9. Use of the collagen derivative according to any one of claims 1 to 3 or the collagen derivative prepared by the preparation method according to any one of claims 4 to 8 in the field of skin care products.

10. A skin care product comprising an effective ingredient and the collagen derivative according to any one of claims 1 to 3 or the collagen derivative prepared by the preparation method according to any one of claims 4 to 8.

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