Micro-crosslinked sodium hyaluronate-polyglutamic acid composite gel and preparation method thereof

Abstract

The invention relates to a micro-crosslinked sodium hyaluronate-polyglutamic acid composite gel and a preparation method thereof. The method comprises the following steps: (1) preparing an enzyme inhibitor solution; (2) preparing a sodium hyaluronate-polyglutamic acid mixed solution; and (3) adding the enzyme inhibitor solution into the sodium hyaluronate-polyglutamic acid mixed solution, stirring and mixing uniformly, and reacting to obtain the micro-crosslinked sodium hyaluronate-polyglutamic acid composite gel. The micro-crosslinked sodium hyaluronate-polyglutamic acid composite gel is formed through interaction of an enzyme inhibitor, sodium hyaluronate and polyglutamic acid. According to the composite gel, the degradation of hyaluronidase is resisted through the enzyme inhibitor, the free radical scavenging capacity of the antioxidant is achieved, and the gel action time is prolonged. Meanwhile, the moisturizing effect of sodium hyaluronate and the repairing effect of water locking of polyglutamic acid have a synergistic effect, so that the composition has a good application prospect and economic value in the field of medical cosmetology.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel and a preparation method thereof. Background Art

[0002] Causes and manifestations of skin aging: Skin aging is a complex process affected by multiple factors. Endogenous aging, such as the decline in physiological functions caused by aging, and exogenous aging, such as ultraviolet rays, environmental pollution and other external stimuli, together promote the gradual aging of the skin. Its external manifestations include thinning, dryness, wrinkles, sagging, pigmentation, etc. of the skin. From a physiological mechanism point of view, the decline in the ability to synthesize collagen, elastin and hyaluronic acid in the skin is a key factor, while the dysfunction of dermal fibroblasts and the reduction of their biosynthetic activity are important attributions to skin aging. In response to this aging mechanism, anti-aging strategies focus on providing a better microenvironment for fibroblasts and resisting oxidative stress. By injecting substances such as sodium hyaluronate, collagen, and antioxidants, the skin condition is improved and the aging process is delayed.

[0003] Applications and Limitations of Sodium Hyaluronate in Anti-Aging: As a mainstream ingredient in anti-aging products currently on the market, hyaluronic acid offers numerous advantages. It is a non-sulfated, long-chain glycosaminoglycan polymer with no species specificity, requiring no skin test prior to injection. It exhibits excellent biocompatibility, is easy to inject, maintains its shape, and does not bind to local cells or cause inflammatory reactions. Its strong water-binding capacity, capable of binding approximately 1,000 times its own weight in water, rapidly fills the skin tissue upon injection into the dermis, producing a cosmetic effect.

[0004] However, hyaluronic acid also has obvious limitations. Its half-life is very short. Under the action of hyaluronidase, long-chain hyaluronic acid will be rapidly degraded into small molecular fragments, enter the circulatory system and be metabolized and absorbed. The degradation process is related to hyaluronidase, temperature, and oxygen free radicals. In order to prolong the duration of action, chemical cross-linking is usually used to prepare hyaluronic acid gel. However, the cross-linking agents 1,4-butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS) commonly used in marketed products are both biologically toxic, and if not completely removed, they may cause adverse reactions such as redness, swelling, and allergies. In addition, the cross-linked sodium hyaluronate has a certain degree of support and is not easy to spread at the injection site, which may form a bulge.

[0005] The potential and advantages of polyglutamic acid in the anti-aging field: As an emerging anti-aging ingredient, polyglutamic acid exhibits strong water-retention capabilities. Experimental data shows that it can prevent water loss in the skin for an extended period of time and is the only known ingredient that can induce a self-increase in the natural moisturizing factor. Regarding whitening, polyglutamic acid offers a safer whitening effect, gently reducing tyrosinase activity in the skin and inhibiting melanin production. From a safety perspective, polyglutamic acid is a natural polymer synthesized by microorganisms with excellent biocompatibility, offering attractive potential for application in the biomedical field. Compared to sodium hyaluronate, polyglutamic acid offers unique advantages in water retention, whitening, and safety, and is expected to become a key development direction in the future anti-aging field.

[0006] In view of this, based on the simulation of proteoglycan and collagen fiber network in skin tissue, a hydrogel with excellent moisturizing and free radical scavenging properties was developed as an anti-aging additive. It can enhance the skin's moisture absorption and moisturizing ability in photoaged areas of the skin, resist skin moisture loss caused by UV, and at the same time scavenge harmful free radicals produced by the body, which is of great significance for delaying and repairing skin aging. Summary of the Invention

[0007] To address the shortcomings of the prior art, the present invention provides a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel and its preparation method. The present invention forms a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel by interacting with an enzyme inhibitor, sodium hyaluronate, and polyglutamic acid. This composite gel resists hyaluronidase degradation through the enzyme inhibitor and the antioxidant's free radical scavenging ability, extending the gel's duration of action. Furthermore, the moisturizing and hydrating effects of sodium hyaluronate and the water-locking and restorative effects of polyglutamic acid work synergistically, giving it promising application prospects and economic value in the field of medical aesthetics.

[0008] The technical solutions of the present invention are as follows:

[0009] A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel comprises the following steps:

[0010] (1) adding enzyme inhibitors to PBS buffer and stirring to mix uniformly to obtain an enzyme inhibitor solution;

[0011] (2) adding sodium hyaluronate to the PBS buffer solution, stirring and mixing uniformly, then sequentially adding polyglutamic acid and an antioxidant, stirring and mixing uniformly, and adjusting the pH to 6 to 8 to obtain a sodium hyaluronate-polyglutamic acid mixed solution;

[0012] (3) Adding the enzyme inhibitor solution obtained in step (1) to the sodium hyaluronate-polyglutamic acid mixed solution obtained in step (2), stirring and mixing uniformly, and sterilizing at 20-30° C. for 5-24 hours to obtain a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel.

[0013] According to the present invention, preferably, in step (1), the enzyme inhibitor is one or more combinations of proanthocyanidins, ellagic acid, and tannic acid.

[0014] Preferably, according to the present invention, in step (1), the mass volume ratio of the enzyme inhibitor and the PBS buffer is (0.05-0.25): (0.5-1), unit: g / mL.

[0015] Preferably, according to the present invention, in step (2), the molecular weight of the sodium hyaluronate is 1 to 2000 kDa; and the polyglutamic acid is γ-polyglutamic acid with a molecular weight of 10 to 300 kDa.

[0016] Preferably, according to the present invention, in step (2), the mass volume ratio of the sodium hyaluronate and the PBS buffer solution is (0.15-0.4): (8-12), unit: g / mL.

[0017] Preferably, according to the present invention, in step (2), the mass ratio of sodium hyaluronate, polyglutamic acid and antioxidant is (0.15-0.4): (0.1-0.4): (0.015-0.05).

[0018] Preferably, according to the present invention, in step (2), the antioxidant is one or more combinations of glutathione, vitamin C, vitamin E and acetylcysteine.

[0019] According to the preferred embodiment of the present invention, in step (2), the pH is adjusted by using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to adjust the solution pH to 6.8-7.2.

[0020] Preferably, according to the present invention, in step (3), the volume of the enzyme inhibitor solution is 0.5 to 3% of the sodium hyaluronate-polyglutamic acid mixed solution.

[0021] The present invention also provides a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel prepared by the above method, and applications of the micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel in the fields of biomedicine, cosmetics, medical cosmetology, and food chemical industry.

[0022] The technical features and beneficial effects of the present invention are as follows:

[0023] 1. The preparation method provided by the present invention uses an enzyme inhibitor as a cross-linking agent. The enzyme inhibitor can undergo micro-cross-linking reactions with polyglutamic acid and sodium hyaluronate, generate interactions, and form various chemical bonds such as hydrogen bonds, ionic bonds, and covalent bonds, thereby preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel. The mechanism of micro-cross-linking between sodium hyaluronate and the enzyme inhibitor is an oxidation-covalent coupling mechanism. Under alkaline conditions, the enzyme inhibitor generates an active intermediate o-quinone\free radical through oxidation, which then undergoes Michael addition reaction or free radical coupling with sodium hyaluronate, so a high concentration of enzyme inhibitor can promote the occurrence of cross-linking reaction. In this method, the enzyme inhibitor simultaneously plays a dual role of inhibiting the degradation of hyaluronic acid and promoting cross-linking. There is no need to add a chemical cross-linking agent with biological toxicity, and it has good biosafety. In addition, the preparation steps are simple, the reaction conditions are mild, and the process is easy to implement.

[0024] 2. The enzyme inhibitors in the micro-crosslinked sodium hyaluronate-polyglutamic acid composite gel of the present invention can inhibit the degradation of sodium hyaluronate by hyaluronidase, while the antioxidants scavenge free radicals and inhibit oxidative stress. The addition of enzyme inhibitors and antioxidants effectively delays the degradation of sodium hyaluronate and polyglutamic acid in the micro-crosslinked sodium hyaluronate-polyglutamic acid composite gel. Furthermore, compared to uncrosslinked sodium hyaluronate solutions, the micro-crosslinked sodium hyaluronate-polyglutamic acid composite gel has a longer duration of action.

[0025] 3. The micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel provided by the present invention not only has excellent moisturizing properties, cell compatibility and free radical scavenging ability, thereby achieving the purpose of resisting skin aging, but also has uses such as facial filling and wrinkle removal, and can be widely used in medical beauty and daily chemical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the free radical scavenging rate of different gels. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with specific drawings and examples. The examples described below are only preferred embodiments of the present invention. It should be noted that the following description is only for the purpose of explaining the present invention and is not intended to limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention. In the following examples, the experimental methods used are conventional methods unless otherwise specified. The reagents used in the examples are all commercially available products.

[0028] Example 1

[0029] A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel comprises the following steps:

[0030] (1) Add 0.25 g of proanthocyanidin and 0.2 mL of 0.1 mol / L sodium hydroxide solution to 0.8 mL of PBS buffer, adjust the pH of the solution to 7.0, and stir until completely dissolved to obtain a proanthocyanidin solution;

[0031] (2) Add 0.15 g of sodium hyaluronate with a molecular weight of 1500 kDa to 10 mL of PBS buffer, stir and mix until completely dissolved, then add 0.2 g of polyglutamic acid with a molecular weight of 40 kDa, stir and mix until completely dissolved, then add 0.015 g of glutathione, stir and mix until completely dissolved, and adjust the pH of the solution to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to obtain a sodium hyaluronate-polyglutamic acid mixed solution;

[0032] (3) Add 100 μL of proanthocyanidin solution to 5 mL of sodium hyaluronate-polyglutamic acid mixed solution, stir and mix evenly, and sterilize at 25°C for 18 h to obtain a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel, which is recorded as gel 1.

[0033] Example 2

[0034] A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel comprises the following steps:

[0035] (1) Add 0.1 g of tannic acid and 0.5 mL of 0.1 mol / L sodium hydroxide solution to 0.5 mL of PBS buffer, adjust the pH of the solution to 7.0, and stir until completely dissolved to obtain a tannic acid solution;

[0036] (2) Add 0.15 g of sodium hyaluronate with a molecular weight of 1500 kDa to 10 mL of PBS buffer, stir and mix until completely dissolved, then add 0.2 g of polyglutamic acid with a molecular weight of 40 kDa, stir and mix until completely dissolved, then add 0.015 g of glutathione, stir and mix until completely dissolved, and adjust the pH of the solution to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to obtain a sodium hyaluronate-polyglutamic acid mixed solution;

[0037] (3) Add 40 μL of tannic acid solution to 5 mL of the sodium hyaluronate-polyglutamic acid mixed solution, stir and mix evenly, and sterilize at 25°C for 5 h to obtain a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel, which is recorded as gel 2.

[0038] Example 3

[0039] A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel comprises the following steps:

[0040] (1) Add 0.1 g of proanthocyanidin and 0.3 mL of 0.1 mol / L sodium hydroxide solution to 0.7 mL of PBS buffer, adjust the pH of the solution to 7.0, and stir until completely dissolved to obtain a proanthocyanidin solution;

[0041] (2) Add 0.2 g of sodium hyaluronate with a molecular weight of 1000 kDa and 0.1 g of sodium hyaluronate with a molecular weight of 200 kDa to 10 mL of PBS buffer, stir and mix until completely dissolved, then add 0.4 g of polyglutamic acid with a molecular weight of 55 kDa, stir and mix until completely dissolved, then add 0.02 g of glutathione, stir and mix until completely dissolved, and adjust the pH of the solution to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to obtain a sodium hyaluronate-polyglutamic acid mixed solution;

[0042] (3) Add 100 μL of proanthocyanidin solution to 5 mL of sodium hyaluronate-polyglutamic acid mixed solution, stir and mix evenly, and sterilize at 25°C for 24 h to obtain a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel, which is recorded as gel 3.

[0043] Example 4

[0044] A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel comprises the following steps:

[0045] (1) Add 0.25 g of tannic acid and 0.5 mL of 0.1 mol / L sodium hydroxide solution to 0.5 mL of PBS buffer, adjust the pH of the solution to 7.0, and stir until completely dissolved to obtain a tannic acid solution;

[0046] (2) Add 0.25 g of sodium hyaluronate with a molecular weight of 800 kDa and 0.15 g of sodium hyaluronate with a molecular weight of 400 kDa to 10 mL of PBS buffer, stir and mix until completely dissolved, then add 0.1 g of polyglutamic acid with a molecular weight of 55 kDa, stir and mix until completely dissolved, then add 0.05 g of glutathione, stir and mix until completely dissolved, and adjust the pH of the solution to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to obtain a sodium hyaluronate-polyglutamic acid mixed solution;

[0047] (3) 40 μL of tannic acid solution was added to 5 mL of the sodium hyaluronate-polyglutamic acid mixed solution, and the mixture was stirred and mixed evenly. After sterilization at 25°C for 20 h, a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel was obtained, which was recorded as gel 4.

[0048] Example 5

[0049] A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel comprises the following steps:

[0050] (1) Add 0.25 g of ellagic acid and 0.5 mL of 0.1 mol / L sodium hydroxide solution to 0.5 mL of PBS buffer, adjust the pH of the solution to 7.0, and stir until the solution is completely dissolved to obtain an ellagic acid solution;

[0051] (2) Add 0.25 g of sodium hyaluronate with a molecular weight of 800 kDa and 0.15 g of sodium hyaluronate with a molecular weight of 400 kDa to 10 mL of PBS buffer, stir and mix until completely dissolved, then add 0.1 g of polyglutamic acid with a molecular weight of 55 kDa, stir and mix until completely dissolved, then add 0.05 g of glutathione, stir and mix until completely dissolved, and adjust the pH of the solution to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to obtain a sodium hyaluronate-polyglutamic acid mixed solution;

[0052] (3) 40 μL of ellagic acid solution was added to 5 mL of the sodium hyaluronate-polyglutamic acid mixed solution, and the mixture was stirred and mixed evenly. After moist heat sterilization at 25°C for 20 h, a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel was obtained, which was recorded as gel 5.

[0053] Comparative Example 1

[0054] 0.15 g of sodium hyaluronate with a molecular weight of 1500 kDa was added to 10 mL of PBS buffer, and the mixture was stirred and mixed until completely dissolved. After sterilization by moist heat, a sodium hyaluronate solution was obtained, which was recorded as gel 6.

[0055] Comparative Example 2

[0056] To 10 mL of PBS buffer, 0.15 g of sodium hyaluronate with a molecular weight of 1500 kDa was added and stirred until completely dissolved. Then, 0.2 g of polyglutamic acid with a molecular weight of 40 kDa was added and stirred until completely dissolved. After wet heat sterilization, a sodium hyaluronate-polyglutamic acid solution was obtained, which was recorded as Gel 7.

[0057] Comparative Example 3

[0058] A method for preparing a micro-cross-linked sodium hyaluronate gel comprises the following steps:

[0059] (1) Add 0.01 g of proanthocyanidin and 0.2 mL of 0.1 mol / L sodium hydroxide solution to 0.8 mL of PBS buffer, adjust the pH of the solution to 7.0, and stir until completely dissolved to obtain a proanthocyanidin solution;

[0060] (2) Add 0.15 g of sodium hyaluronate with a molecular weight of 1500 kDa to 10 mL of PBS buffer, stir and mix until completely dissolved, then add 0.015 g of glutathione, stir and mix until completely dissolved, and adjust the pH of the solution to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to obtain a sodium hyaluronate mixed solution.

[0061] (3) Add 100 μL of proanthocyanidin solution to 5 mL of the sodium hyaluronate mixed solution, stir and mix evenly, and sterilize at 25°C for 18 h to obtain a slightly cross-linked sodium hyaluronate gel, which is recorded as gel 8.

[0062] Comparative Example 4

[0063] A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel comprises the following steps:

[0064] (1) Add 0.25 g of tannic acid and 0.5 mL of 0.1 mol / L sodium hydroxide solution to 0.5 mL of PBS buffer, adjust the pH of the solution to 7.0, and stir until completely dissolved to obtain a tannic acid solution;

[0065] (2) Add 0.25 g of sodium hyaluronate with a molecular weight of 800 kDa and 0.15 g of sodium hyaluronate with a molecular weight of 400 kDa to 10 mL of PBS buffer, stir and mix until completely dissolved, then add 0.1 g of polyglutamic acid with a molecular weight of 55 kDa, stir and mix until completely dissolved, then add 0.005 g of glutathione, stir and mix until completely dissolved, and adjust the pH of the solution to 7.0 using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to obtain a sodium hyaluronate-polyglutamic acid mixed solution;

[0066] (3) 40 μL of tannic acid solution was added to 5 mL of the sodium hyaluronate-polyglutamic acid mixed solution, and the mixture was stirred and mixed evenly. After sterilization at 25°C for 20 h, a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel was obtained, which was recorded as gel 9.

[0067] Test Example 1

[0068] Gels 1, 3, 6 to 8 prepared in Example 1, Example 3, and Comparative Examples 1 to 3 were placed in 10 U / mL, 30 U / ml, and 60 U / ml hyaluronidase solutions, respectively, for accelerated degradation. The degradation rates of the corresponding gels were measured after 8 h, 24 h, and 48 h, respectively. The results are shown in Table 1 below.

[0069] Table 1

[0070]

[0071] As shown in Table 1, gels 1 and 3 prepared in Examples 1 and 3 exhibited far superior resistance to enzymatic degradation than gels 6 to 8 prepared in Comparative Examples 1 to 3. This demonstrates that the present invention effectively produces enzymatically resistant micro-crosslinked sodium hyaluronate-polyglutamic acid composite gels by using enzyme inhibitors as crosslinking agents to interact with polyglutamic acid and sodium hyaluronate to form various chemical bonds, including hydrogen bonds, ionic bonds, and covalent bonds.

[0072] Test Example 2

[0073] 0.05 g samples of gels 1, 3, and 6 to 8 prepared in Example 1, Example 3, and Comparative Examples 1 to 3 were respectively weighed and placed in a weighing bottle with a diameter of 30 mm. The samples were diluted to 10% with deionized water and placed in a desiccator with a relative humidity of 43% and an ambient temperature of 20°C. The mass changes of the samples were measured after standing for 6 h, 16 h, 24 h, and 48 h.

[0074] The formula for calculating the moisturizing rate is: moisturizing rate = (1-(M1-M0) / M1)×100%.

[0075] Where M0 represents the initial mass of the gel (g), and M1 represents the mass of the gel after a certain period of time (g). A higher moisture retention rate indicates better moisturizing performance. Each moisture absorption and moisturizing experiment was repeated three times, and the average results were taken. Table 2 shows the results below.

[0076] Table 2

[0077]

[0078]

[0079] As shown in Table 2, the moisturizing ability of composite gel 1 and composite gel 3 prepared in Examples 1 and 3 is significantly better than that of gels 6 to 8 prepared in Comparative Examples 1 to 3, which indicates that the product provided by the present invention has good moisturizing ability.

[0080] Test Example 3

[0081] Weigh 8 mg of DPPH and place it in a 100 ml brown volumetric flask. Dissolve it by ultrasonication and then adjust the volume to the mark to obtain a DPPH solution. Chop the gels 2, 4, 6-7, and 9 prepared in Examples 2 and 4 and Comparative Examples 1-2 and 4 into small pieces and dissolve them in 30% ethanol for 1.5 hours. Then ultrasonicate them for 30 minutes and centrifuge to obtain the supernatant. Take 2 mL of each of the gel solutions 2, 4, 6-7, and 9 and place them in a test tube. Add 2 mL of DPPH solution respectively, mix well, and then protect from light for 30 minutes. Measure the absorbance at a wavelength of 517 nm and calculate the free radical scavenging rate of different gels. The results are as follows: Figure 1 shown.

[0082] The calculation formula is as follows: Clearance % = (1-(A s -A b ) / A c )×100%.

[0083] Among them, A s is the absorbance value of gel solution; A b is the absorbance value when DPPH solution is replaced with anhydrous ethanol; A c The absorbance value is when the gel solution is replaced with water.

[0084] Depend on Figure 1 It can be seen that the free radical scavenging ability of gels 2 and 4 prepared in Examples 2 and 4 is significantly greater than that of gels 6 to 7 and 9 prepared in Comparative Examples 1 to 2 and 4. This demonstrates that the enzyme inhibitors and antioxidants in the micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel of the present invention synergistically exert free radical scavenging effects. When the amount of glutathione is reduced, the free radical scavenging rate decreases significantly, and the example gels provided by the present invention have good antioxidant capacity.

[0085] Test Example 4

[0086] The gels prepared in Examples 1-5 were coated onto 10 mm x 10 mm filter paper as a support, and the gel solutions were used as test groups. The positive group consisted of natural latex, and the negative group consisted of high-density polyethylene (HDPE), with three replicates for each group. Each of the three solutions was overlaid onto a layer of L929 cells and incubated in a 37°C, 5% CO2 incubator for 48 hours. The plates were then removed and the sample locations marked on the bottom with a marker. The samples were discarded and the culture medium removed. 2 mL of neutral red was added to each plate and incubated for 1 hour. The neutral red was then aspirated and discarded, and 2 mL of PBS buffer was added. Cell morphology was observed microscopically. Cytotoxicity was expressed using a reaction grade (Rate of Response). The results are shown in Table 3 below.

[0087] Table 3

[0088]

[0089]

[0090] As shown in Table 3, the cells in the experimental group showed no denaturation or lysis, and no cytotoxicity was observed. This indicates that the micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel provided by the present invention not only has excellent resistance to enzymatic degradation, moisture retention, and free radical scavenging ability, but also has good cell compatibility, and can be widely used in fields such as medical aesthetics and daily chemicals.

[0091] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel, characterized in that: The steps are as follows: (1) adding enzyme inhibitors to PBS buffer and stirring to mix uniformly to obtain an enzyme inhibitor solution; (2) adding sodium hyaluronate to the PBS buffer solution, stirring and mixing uniformly, then sequentially adding polyglutamic acid and an antioxidant, stirring and mixing uniformly, and adjusting the pH to 6 to 8 to obtain a sodium hyaluronate-polyglutamic acid mixed solution; (3) Adding the enzyme inhibitor solution obtained in step (1) to the sodium hyaluronate-polyglutamic acid mixed solution obtained in step (2), stirring and mixing uniformly, and sterilizing at 20-30° C. for 5-24 hours to obtain a slightly cross-linked sodium hyaluronate-polyglutamic acid composite gel.

2. The preparation method according to claim 1, wherein In step (1), the enzyme inhibitor is one or more combinations of proanthocyanidins, ellagic acid, and tannic acid.

3. The preparation method according to claim 1, wherein In step (1), the mass volume ratio of the enzyme inhibitor and PBS buffer is (0.05-0.25): (0.5-1), unit: g / mL.

4. The preparation method according to claim 1, wherein In step (2), the molecular weight of the sodium hyaluronate is 1 to 2000 kDa; the polyglutamic acid is γ-polyglutamic acid with a molecular weight of 10 to 300 kDa; the mass-to-volume ratio of the sodium hyaluronate to the PBS buffer is (0.15 to 0.4): (8 to 12), unit: g / mL.

5. The preparation method according to claim 1, wherein In step (2), the mass ratio of sodium hyaluronate, polyglutamic acid and antioxidant is (0.15-0.4): (0.1-0.4): (0.015-0.05).

6. The preparation method according to claim 1, wherein In step (2), the antioxidant is one or more combinations of glutathione, vitamin C, vitamin E and acetylcysteine.

7. The preparation method according to claim 1, wherein In step (2), the pH is adjusted by using 0.1 mol / L sodium hydroxide solution or 0.1 mol / L hydrochloric acid solution to adjust the solution pH to 6.8-7.

2.

8. The preparation method according to claim 1, wherein In step (3), the volume of the enzyme inhibitor solution is 0.5 to 3% of the sodium hyaluronate-polyglutamic acid mixed solution.

9. Micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel prepared by the method according to any one of claims 1 to 9.

10. Use of the micro-cross-linked sodium hyaluronate-polyglutamic acid composite gel according to claim 9 in the fields of biomedicine, cosmetics, medical cosmetology and food chemical industry.

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