Collagen-hyaluronic acid or hyaluronate composite solution

Abstract

The invention relates to the technical field of biological products, in particular to a collagen-hyaluronic acid or hyaluronate composite solution. By controlling the concentration of the collagen concentrated solution, the concentration of the hyaluronic acid / hyaluronate solution and the mass ratio of the collagen to the hyaluronic acid / hyaluronate in the composite solution, the composite solution has good storage stability and whiteness and is easy to push. The composite solution is safe to use, simple in preparation method and easy to industrialize, can be used in the field of injection medical treatment or injection filling, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to a collagen-hyaluronic acid or hyaluronate composite solution. Background Art

[0002] As we age, collagen in the skin begins to deplete, leading to various issues such as sagging, dullness, and pigmentation. Whitening, filling, and moisturizing have become increasingly sought-after beauty products. Collagen inhibits the production of melanin synthase and helps disperse melanin particles, preventing them from clumping and forming dark spots. Furthermore, collagen accelerates skin cell turnover, causing the loss of old, pigmented cells, contributing to a more even and fair complexion. In summary, collagen's ability to improve skin hydration and elasticity can indirectly affect skin whitening. Hyaluronic acid (HA), or hyaluronate, can be categorized by molecular weight as large, medium, or small molecule HA or HA, each with its own distinct effects. For example, the molecular weight of large-molecule sodium hyaluronate ranges from 1800 KDa to 2200 KDa, and it has viscoelasticity, wettability and moisturizing properties, forming a protective film on the surface of the skin; the molecular weight of medium-molecule sodium hyaluronate ranges from 1000 KDa to 1800 KDa, and it can form a breathable film on the surface of the skin to lock in moisture, and is mainly used for moisturizing; the molecular weight of small-molecule sodium hyaluronate ranges from 10 KDa to 1000KDa, and it is easily absorbed by the skin and can participate in the internal metabolism of the skin.

[0003] Collagen can inhibit melanin formation, make the skin fairer, and play the role of skin whitening and tightening; Hyaluronic acid substances mainly provide a hydration environment for cells, focus on replenishing water, play the role of moisturizing and lubricating, and both have their advantages and disadvantages. If collagen and hyaluronic acid substances are compounded, both deficiencies can be made up, and great development space is provided for the development of the beauty field. For example, prior art CN 114796011 A discloses a composite solution and its preparation process for the combined application of recombinant type III humanized collagen and hyaluronic acid, which adopts sodium hyaluronate as a filling support, and its water absorption and moisture retention performance is excellent. After injection, the effect of moisturizing and anti-wrinkle is immediately achieved, and then the space occupancy effect of substances such as recombinant type III humanized collagen and glutathione is stimulated to synthesize collagen by the body itself, while tranexamic acid is used to play the role of anti-allergic and anti-inflammatory. However, the collagen-hyaluronic acid or hyaluronate composite solution in the above-mentioned prior art is not good in long-term stability. Other publicly reported collagen-hyaluronate composite solutions also have the defect that whiteness is not enough or is not easy to push.

[0004] Therefore, there is an urgent need in this field to develop a collagen-hyaluronic acid / hyaluronate composite solution with higher whiteness, better long-term stability and easy pushing. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a collagen-hyaluronic acid / hyaluronate composite solution, whose raw materials include collagen concentrate and hyaluronic acid / hyaluronate solution; the concentration of the collagen concentrate is 20-40 mg / mL; the concentration of the hyaluronic acid / hyaluronate solution is 8-12 mg / mL; in the composite solution, the mass ratio of collagen to hyaluronic acid / hyaluronate is (0.5-1.5):1.

[0006] The present invention has found that by controlling the concentration of the collagen concentrate, the concentration of the hyaluronic acid / hyaluronate solution, and the final mass ratio of collagen to hyaluronic acid / hyaluronate within the above range, the composite solution system can be stored for a long time in a low temperature environment (2°C~10°C). For at least 60 days, the composite solution system can still remain stable without phase separation. At the same time, the composite solution system has good whiteness and is easy to push.

[0007] Preferably, the concentration of the collagen concentrate is 20-30 mg / mL; the concentration of the hyaluronic acid / hyaluronate solution is 8-10 mg / mL; and in the composite solution, the mass ratio of collagen to hyaluronic acid / hyaluronate is (0.6-1.25):1.

[0008] At the above mass ratio, the composite solution system is easier to push.

[0009] More preferably, the concentration of the collagen concentrate is 28-30 mg / mL; the concentration of the hyaluronic acid / hyaluronate solution is 8-10 mg / mL; and in the composite solution, the mass ratio of collagen to hyaluronic acid / hyaluronate is (1-1.25):1.

[0010] At the above mass ratio, the composite solution system not only has a small pushing force but also has good whiteness, and can balance the pushing force and whiteness.

[0011] The composite solution of the present invention has a stable compatibility of more than 60 days; and / or the degree of self-assembly of collagen in the composite solution is more than 98% (for example, more than 98%, 99%); and / or the pushing force in the syringe of the composite solution is less than 5.5N when tested using a 30G injection needle; and / or the cell survival rate of the composite solution is more than 100%; and / or the viscosity of the composite solution is 199-255 mPa.s.

[0012] Preferably, the composite solution has a pushing force in a syringe of 4.5 to 5.5 N (e.g., 4.5 N, 4.6 N, 4.7 N, 4.8 N, 4.9 N, 5.0 N, 5.1 N, 5.2 N, 5.3 N, 5.4 N, or 5.5 N) when tested using a 30G injection needle.

[0013] Preferably, the pH of the composite solution is 6.0-8.0, and the osmotic pressure is 250 mOsm / kg-280 mOsm / kg.

[0014] Preferably, the collagen concentrate is a concentrate of the collagen solution after salting out.

[0015] Preferably, the collagen concentrate is a concentrate obtained by mixing a hydrochloric acid solution of collagen with a saline solution and then subjecting the mixture to salting-out.

[0016] More preferably, the collagen concentrate is a concentrate obtained by mixing a hydrochloric acid solution of collagen with a saline solution, adjusting the pH to 6.0-8.0, and salting out at 30° C.-37° C.

[0017] Preferably, the concentration of collagen in the collagen solution is 1.5 to 6.0 mg / mL (preferably 1.5 to 3 mg / mL).

[0018] Preferably, the pH of the hydrochloric acid solution is 2.0 to 5.0 (preferably 2.0 to 3.0).

[0019] Preferably, the salt solution in the salting out is an aqueous solution of disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride; or the salt solution is an aqueous solution of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium chloride; or the salt solution is an aqueous solution of disodium hydrogen phosphate, sodium dihydrogen phosphate and potassium chloride; or the salt solution is an aqueous solution of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride.

[0020] More preferably, the salt solution in the salting out is an aqueous solution of 0.4% to 0.5% (w / w) disodium hydrogen phosphate, 0.02% to 0.06% (w / w) sodium dihydrogen phosphate and 0.6% to 0.8% (w / w) sodium chloride; or the salt solution is an aqueous solution of 0.1% to 0.2% (w / w) dipotassium hydrogen phosphate, 0.03% to 0.07% (w / w) potassium dihydrogen phosphate and 0.8% to 1.2% (w / w) potassium chloride. aqueous solution; or the saline solution is an aqueous solution of 0.1%~0.3% (w / w) disodium hydrogen phosphate, 0.01%~0.05% (w / w) sodium dihydrogen phosphate and 0.8%~1.2% (w / w) potassium chloride; or the saline solution is an aqueous solution of 0.1%~0.3% (w / w) dipotassium hydrogen phosphate, 0.03%~0.07% (w / w) potassium dihydrogen phosphate and 0.6%~0.8% (w / w) sodium chloride.

[0021] More preferably, the salt solution in the salting-out is a PBS solution.

[0022] Preferably, the concentration of the salt solution in the salting-out is 0.08 M to 0.20 M, more preferably 0.1 M to 0.15 M.

[0023] When the concentration of the salt solution is controlled within the above range, the compatibility of the system is better and the triple helix structure of collagen is completely retained.

[0024] Preferably, the molecular weight of the hyaluronic acid / hyaluronate is 1500-2000 KDa (preferably 1500-1600 KDa).

[0025] Preferably, the hyaluronic acid / hyaluronate solution is a PBS solution of hyaluronic acid / hyaluronate.

[0026] Preferably, the hyaluronic acid / hyaluronate solution is a mixture of hyaluronic acid / hyaluronate and 0.01-0.05 M PBS.

[0027] Preferably, the hyaluronate is sodium hyaluronate, calcium hyaluronate, potassium hyaluronate or magnesium hyaluronate.

[0028] Furthermore, the present invention provides a method for preparing the composite solution, comprising: mixing a collagen concentrate with a hyaluronic acid / hyaluronate solution, and homogenizing to obtain the composite solution.

[0029] Preferably, the collagen concentrate is mixed with the hyaluronic acid / hyaluronate solution in a volume ratio of 1:(2-8).

[0030] Preferably, the preparation method comprises: mixing a salt solution and a collagen solution at 2°C to 10°C, adjusting the pH of the system to 6.0-8.0, stirring at 30°C to 37°C, and concentrating to obtain a collagen concentrate; mixing a hyaluronic acid / hyaluronate solution with the collagen concentrate and homogenizing to obtain the composite solution.

[0031] Preferably, the pH of the system is adjusted to 6.8-7.2.

[0032] Preferably, the collagen solution is a hydrochloric acid solution of collagen.

[0033] Preferably, the mass ratio of the saline solution to the collagen solution is 1:(8-12).

[0034] Preferably, the composite solution is prepared by mixing the hyaluronic acid / hyaluronate solution with the collagen concentrate and homogenizing to remove large particles.

[0035] Preferably, the homogenization speed is above 13000 rpm.

[0036] Preferably, the homogenization is cycled 3 to 6 times at 2° C. to 10° C.

[0037] Furthermore, the present invention provides a product containing the composite solution; the product is a cosmetic or a beauty product.

[0038] During specific implementation, the product also includes auxiliary materials acceptable in the field of cosmetics or beauty products.

[0039] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a collagen-hyaluronic acid / hyaluronate composite solution with excellent storage stability. By controlling the concentration of the collagen concentrate, the concentration of the hyaluronic acid / hyaluronate solution, and the mass ratio of collagen to hyaluronic acid / hyaluronate in the composite solution, the composite solution exhibits excellent storage stability, whiteness, and ease of squeezing. The composite solution of the present invention is safe to use, has a simple preparation method, and is readily industrializable. It can be used in the fields of injectable medical treatment or injectable filling, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a photograph of the composite solution of Example 1.

[0041] Figure 2 This is the electrophoresis diagram of the composite solution of Example 1.

[0042] Figure 3 1 is the circular dichroism spectrum of the composite solution of Example 1. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, those without specifying specific techniques or conditions are described in accordance with the techniques or conditions described in the literature in this area, or are carried out according to product specifications. Reagents or instruments used are not specified by manufacturer and are conventional products that can be purchased through regular channels.

[0044] Examples 1 to 5 This embodiment provides a collagen-hyaluronate composite solution, the preparation method of which is as follows: (1) Weigh dry type I collagen (Yantai Desheng Marine Biotechnology Co., Ltd.) and dissolve it in a hydrochloric acid solution with a pH of 2.0 at 4°C to a concentration of 1.5 mg / mL. Then prepare a 0.11 M PBS solution and add the PBS solution to the collagen solution at a ratio of 1 / 10 (mass ratio of PBS solution to collagen solution). Adjust the pH of the solution to 7.0 and place it at 37°C with constant stirring for 24 h to obtain a collagen neutralization solution. Then, centrifuge at 11,000 rpm and 4°C for 15 min and concentrate to obtain a collagen concentrate.

[0045] (2) Take an appropriate amount of sodium hyaluronate powder (molecular weight 1500 KDa) at room temperature and dissolve it in 0.01 M PBS to prepare sodium hyaluronate solutions with different initial concentrations.

[0046] (3) Collagen concentrate and sodium hyaluronate solution were mixed in a volume ratio of 1:3, as shown in Table 1; the mixed solution was homogenized at 13,000 rpm to remove large particles, and the homogenization cycle was repeated 3 times at 4°C to obtain a collagen-sodium hyaluronate composite solution, which was then filled into a prefilled syringe.

[0047] Table 1

[0048] Test Example 1 The properties of the collagen-sodium hyaluronate composite solutions prepared in the above examples and comparative examples were tested and characterized. The specific test methods are as follows: (1) Appearance: Observe the state and color of the composite solution at the time of preparation and after storage at 2℃~10℃ for 60 days with naked eyes. For the convenience of statistics, the shades of white are defined here, from lightest to darkest, as transparent, slightly white, light white, white, and milky white.

[0049] (2) Collagen self-assembly degree: This is defined as the percentage of collagen molecules that form collagen fibers. A small amount of collagen neutralization solution was centrifuged at 14,000 rpm for 20 min. The supernatant was collected and the collagen content in the supernatant was determined using a protein quantitative analysis kit. The collagen self-assembly degree (%) was calculated according to the following formula: (C0-C0) / C0×100%, where C0 is the initial concentration of the collagen solution (mg / mL) and C is the protein concentration in the supernatant of the collagen neutralization solution (mg / mL).

[0050] (3) Viscosity: At 25°C, the rheometer was used to measure the viscosity of the sample at a shear rate of 0.001 s -1 ~1000 s -1 Flow scanning was performed under the condition of 158.6 s -1The viscosity data corresponding to this specific shear rate are compared and analyzed.

[0051] (4) Stability test: In order to investigate the stability of the composite solution, the collagen-sodium hyaluronate composite solution was placed in a refrigerator at 2°C to 10°C, and the appearance was observed after 60 days.

[0052] (5) Pushing force test: The collagen-sodium hyaluronate composite solution was equilibrated at room temperature for 1 h, and the push rod and 30G injection needle were installed. A small amount of air was removed from the front end of the syringe and the syringe was installed on the testing equipment. The test distance was set to full scale and the pushing speed was set to 10 mm / min to 30 mm / min. The pushing force was measured using a universal material testing machine.

[0053] The test results are shown in Table 2. The composite solution of Example 1 is shown in Table 2. Figure 1 shown.

[0054] The composite solution in the example exhibited excellent extrusion performance, with a smooth and easy extrusion process. Furthermore, no needle clogging occurred after three consecutive injections. These results demonstrate that the sample in the example is relatively evenly dispersed, making it suitable for use as a medical aesthetic injection, significantly reducing the difficulty of the injection procedure.

[0055] Table 2

[0056] Test Example 2 Other properties of the collagen-sodium hyaluronate composite solution prepared in the above example were tested and characterized. The specific test methods are as follows: (1) Cytotoxicity test: Mouse fibroblasts (L929) were selected and the cell suspension was diluted with RPMI1640 medium to a cell density of 10 4 100 μL of cell suspension (10 4 ) were pre-incubated in an incubator for 24 hours to allow the cells to adhere and grow. The upper culture medium was then aspirated, and 100 μL of the test sample was added to the experimental group, 100 μL of sterile water was added to the negative control group, and 100 μL of culture medium was added to the positive control group. The cells were incubated for another 24 hours. The supernatant was then aspirated, and 100 μL of MTT solution was added. The cells were incubated in the dark for 4 hours. The supernatant was removed, and finally 100 μL of DMSO was added. The absorbance at 570 nm was measured. The relative cell viability was calculated according to the following formula: Among them, OD t ,OD - ,OD + are the absorbance at 570 nm of the test group, negative group, and positive group, respectively.

[0057] The test results are shown in Table 3.

[0058] Table 3

[0059] In the cytotoxicity test, the relative cell survival rates of the composite solutions of all examples were above 100%, which showed good biosafety and minimized the potential cytotoxicity risk.

[0060] (2) Collagen identification test: Dilute the sample with purified water to a solution with a collagen concentration of 1 mg / mL to 2 mg / mL. Follow the method specified in Method 5 of Electrophoresis in 0541 of the Pharmacopoeia of the People's Republic of China (Volume IV) (2020 edition), using SDS-polyacrylamide gel electrophoresis, with a separation gel concentration of 10% and a sample load of not less than 20 μg. Compare the bands of the sample and type I collagen standard after SDS-polyacrylamide gel electrophoresis analysis.

[0061] The electrophoresis results are as follows Figure 2 As shown, the electrophoresis pattern of the collagen-sodium hyaluronate composite solution in Example 1 is consistent with that of type I collagen, and the other examples also have substantially identical electrophoresis patterns. Specifically, two bands appear near the molecular weight range of 100-135 kDa, corresponding to the α1 and α2 chains of the collagen molecule, respectively; while a single band appears near the molecular weight range of approximately 245 kDa, corresponding to the β chain of the collagen molecule.

[0062] (3) Circular dichroism test: A 0.2 mg / mL solution was prepared and injected into a quartz sample cell. The CD of the sample of Example 1 was measured using a circular dichroism spectrometer.

[0063] CD spectrum Figure 3 As shown, other embodiments also have substantially the same CD spectra. Specifically, positive and negative absorption peaks appear between 195 nm and 240 nm.

[0064] The results of the above-mentioned collagen identification test and circular dichroism test show that in the collagen-sodium hyaluronate composite solution, the collagen molecules still retain the triple helix structure. The retention of this structure means that the composite solution has biological activity and has potential application value in the fields of biomedical materials and so on.

[0065] Examples 6 to 9 This embodiment provides a collagen-hyaluronate composite solution, and the preparation method is different from that of Example 1 only in that the saline solution (PBS solution) in step (1) is replaced by the saline solutions in Table 4.

[0066] Table 4

[0067] The performance was tested according to the test method of Test Example 1, and the test results are shown in Table 5.

[0068] Table 5

[0069] Examples 10-12 This embodiment provides a collagen-hyaluronate composite solution. The preparation methods of Examples 10 to 12 are respectively different from those of Examples 1 to 3 in that the molecular weight of the sodium hyaluronate powder is 2000 KDa.

[0070] The performance was tested according to the test method of Test Example 1, and the test results are shown in Table 6.

[0071] Table 6

[0072] Finally, 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A collagen-hyaluronic acid / hyaluronate composite solution, characterized in that: Its raw materials include collagen concentrate and hyaluronic acid / hyaluronate solution; the concentration of the collagen concentrate is 20-40 mg / mL; the concentration of the hyaluronic acid / hyaluronate solution is 8-12 mg / mL; in the composite solution, the mass ratio of collagen to hyaluronic acid / hyaluronate is (0.5-1.5):

1.

2. The composite solution according to claim 1, characterized in that The concentration of the collagen concentrate is 20-30 mg / mL; the concentration of the hyaluronic acid / hyaluronate solution is 8-10 mg / mL; in the composite solution, the mass ratio of collagen to hyaluronic acid / hyaluronate is (0.6-1.25):

1.

3. The composite solution according to claim 2, characterized in that The concentration of the collagen concentrate is 28-30 mg / mL; the concentration of the hyaluronic acid / hyaluronate solution is 8-10 mg / mL; in the composite solution, the mass ratio of collagen to hyaluronic acid / hyaluronate is (1-1.25):

1.

4. The composite solution according to any one of claims 1 to 3, characterized in that The composite solution has a stable compatibility of more than 60 days; and / or, when the composite solution is tested using a 30G injection needle, the pushing force in the syringe is less than 5.5N.

5. The composite solution according to any one of claims 1 to 3, characterized in that The degree of self-assembly of collagen in the composite solution is greater than 98%; and / or the cell survival rate of the composite solution is greater than 100%; and / or the viscosity of the composite solution is 199-255 mPa.s.

6. The composite solution according to any one of claims 1 to 3, characterized in that The collagen concentrate is a concentrate of the collagen solution after salting out; Preferably, the collagen concentrate is a concentrate obtained by mixing a hydrochloric acid solution of collagen with a saline solution and then subjecting it to salting out; Preferably, the concentration of collagen in the collagen solution is 1.5 to 6.0 mg / mL; Preferably, the pH of the hydrochloric acid solution is 2.0-5.

0.

7. The composite solution according to claim 6, characterized in that The salt solution in the salting out is an aqueous solution of disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride; or the salt solution is an aqueous solution of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium chloride; or the salt solution is an aqueous solution of disodium hydrogen phosphate, sodium dihydrogen phosphate and potassium chloride; or the salt solution is an aqueous solution of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride; Preferably, the salt solution in the salting out is an aqueous solution of 0.4% to 0.5% (w / w) disodium hydrogen phosphate, 0.02% to 0.06% (w / w) sodium dihydrogen phosphate and 0.6% to 0.8% (w / w) sodium chloride; or the salt solution is an aqueous solution of 0.1% to 0.2% (w / w) dipotassium hydrogen phosphate, 0.03% to 0.07% (w / w) potassium dihydrogen phosphate and 0.8% to 1.2% (w / w) potassium chloride. aqueous solution; or the saline solution is an aqueous solution of 0.1%~0.3% (w / w) disodium hydrogen phosphate, 0.01%~0.05% (w / w) sodium dihydrogen phosphate and 0.8%~1.2% (w / w) potassium chloride; or the saline solution is an aqueous solution of 0.1%~0.3% (w / w) dipotassium hydrogen phosphate, 0.03%~0.07% (w / w) potassium dihydrogen phosphate and 0.6%~0.8% (w / w) sodium chloride.

8. The composite solution according to any one of claims 1 to 3, characterized in that The molecular weight of the hyaluronic acid / hyaluronate is 1500-2000 KDa, preferably 1500-1600 KDa.

9. The method for preparing the composite solution according to any one of claims 1 to 8, characterized in that: include: Mixing the collagen concentrate with the hyaluronic acid / hyaluronate solution and homogenizing to obtain the composite solution; Preferably, the preparation method comprises: mixing a salt solution and a collagen solution at 2°C to 10°C, adjusting the pH of the system to 6.0-8.0, stirring at 30°C to 37°C, and concentrating to obtain a collagen concentrate; mixing a hyaluronic acid / hyaluronate solution with the collagen concentrate and homogenizing to obtain the composite solution; Preferably, the pH of the system is adjusted to 6.8-7.2; Preferably, the mass ratio of the saline solution to the collagen solution is 1:(8-12).

10. A product, characterized in that The product contains the composite solution according to any one of claims 1 to 8 or the composite solution prepared by the preparation method according to claim 9; and the product is a cosmetic or a beauty product.

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