Antioxidant composition as well as preparation method and application thereof

Abstract

The invention relates to an antioxidant composition as well as a preparation method and application thereof. The antioxidant composition comprises nano cross-linked sodium hyaluronate and an antioxidant, the nano cross-linked sodium hyaluronate comprises second sodium hyaluronate cross-linked by cross-linked sodium hyaluronate, and the cross-linked sodium hyaluronate comprises first sodium hyaluronate cross-linked by modified chitosan; wherein the modified chitosan comprises a deacetylated chitosan main chain, the main chain is grafted with a group through an ether bond, and p is an integer between 0 and 200. The antioxidant composition provided by the invention can well permeate through skin, and has a wide application prospect in the field of skin care products.

Description

Technical Field

[0001] The present invention relates to an antioxidant composition, a preparation method and application thereof. Background Art

[0002] The process of skin aging is mainly due to environmental factors, and the free radicals caused by the oxidative stress reaction caused by factors such as sunlight exposure, radiation, pressure are produced in a large number, causing the change of skin structure, finally causing the collapse of the structure of dermis, forming wrinkles. Therefore how to reduce the generation of excessive free radicals, and remove the damage to skin, for anti-aging play a vital role. In this area, there are a large amount of antioxidants, such as bosaicin, ergothioneine, ectoine etc., which are used in anti-aging products, and have played a significant anti-aging effect.

[0003] However, the efficiency of antioxidants entering the skin is relatively low, which greatly reduces the anti-aging effect of antioxidants. Therefore, how to improve their penetration efficiency is of great significance for the anti-aging effect of antioxidants. At present, in order to improve the efficiency of transdermal absorption, it is generally achieved by adding small molecule transdermal penetration enhancers, including ethanol, water-soluble azone, etc. However, these small molecules mainly disrupt or destroy the structure of the skin's stratum corneum, allowing antioxidants to enter the skin. However, this method often causes damage to the skin barrier and ultimately leads to skin sensitivity. Therefore, whether it is possible to achieve transdermal absorption and high efficiency of antioxidants without damaging the stratum corneum or in a state where the skin structure can be restored has important practical significance. Summary of the Invention

[0004] In order to solve the problem of low absorption efficiency, the present invention provides an antioxidant composition, a preparation method and application thereof.

[0005] In a first aspect, the present invention provides an antioxidant composition comprising nano-cross-linked sodium hyaluronate and an antioxidant.

[0006] The nano-cross-linked sodium hyaluronate includes a second sodium hyaluronate cross-linked by cross-linked sodium hyaluronate, and the cross-linked sodium hyaluronate includes a first sodium hyaluronate cross-linked by modified chitin;

[0007] Wherein, the modified chitosan comprises a chitosan backbone, and the backbone is grafted via an ether bond. group, p is an integer between 0 and 200.

[0008] The inventors of the present application unexpectedly discovered that by using nano-cross-linked sodium hyaluronate as a penetration enhancer, the water content of the skin's stratum corneum is increased by increasing the water content on the skin surface, thereby increasing the hydration of the skin's stratum corneum, and enhancing the absorption of antioxidants through the skin's encapsulation effect, thereby increasing the anti-aging effect of antioxidants.

[0009] Furthermore, the present invention obtains modified chitosan by grafting ethylene oxide groups containing or not polyethylene glycol or grafting propylene oxide groups containing or not polyethylene glycol onto deacetylated chitosan. After the modified chitosan is cross-linked with sodium hyaluronate, it can be used as a viscosity modifier to significantly reduce the viscosity of sodium hyaluronate. After the modified chitosan cross-linked sodium hyaluronate is mixed with sodium hyaluronate again, nano-sodium hyaluronate with significantly reduced viscosity is obtained, which can effectively increase the water content of the skin, thereby hydrating the stratum corneum of the skin, thereby increasing the permeability of the skin, and ultimately achieving the effect of bringing the efficacy substance into the skin, increasing the amount of drug entering the skin, and improving the bioavailability of the efficacy substance.

[0010] According to some embodiments of the present invention, p is an integer between 0 and 200, for example, 0, 1, 2, 3, 4, 5, 8, 10, 20, 25, 30, 50, 80, 100, 120, 150, 180, 200 or any value therebetween.

[0011] According to some embodiments of the present invention, p is an integer between 1 and 200. According to some embodiments of the present invention, p is an integer between 1 and 100. According to some embodiments of the present invention, p is an integer between 1 and 20. According to some embodiments of the present invention, p is an integer between 3 and 20.

[0012] According to some embodiments of the present invention, the weight ratio of the nano-cross-linked sodium hyaluronate and the antioxidant is 1:(1-50), for example, 1:1, 1:5, 1:8, 1:10, 1:12, 1:15, 1:17, 1:20, 1:23, 1:25, 1:27, 1:30, 1:35, 1:40 or any value therebetween.

[0013] According to some embodiments of the invention, the antioxidant is selected from one or more of ergothioneine, bosine or ectoine.

[0014] According to some embodiments of the present invention, the nano-cross-linked sodium hyaluronate and the antioxidant are selected from one or more of ergothioneine, bosine or ectoine to form nanoparticles.

[0015] In some embodiments, the method for preparing the modified chitin comprises the following steps:

[0016] The modified chitosan is obtained by reacting deacetylated chitosan with an epoxidation agent; wherein the epoxidation agent comprises epichlorohydrin and / or chlorinated polyethylene glycol grafted with ethylene oxide.

[0017] In some embodiments, the degree of polymerization of the ethylene oxide grafted chlorinated polyethylene glycol is 1 to 200, for example, 1, 2, 3, 4, 5, 8, 10, 20, 25, 30, 50, 80, 100, 120, 150, 180, 200 or any value therebetween. In some embodiments, the degree of polymerization of the ethylene oxide grafted chlorinated polyethylene glycol is 1 to 100. In some embodiments, the degree of polymerization of the ethylene oxide grafted chlorinated polyethylene glycol is 1 to 20. In some embodiments, the degree of polymerization of the ethylene oxide grafted chlorinated polyethylene glycol is 3 to 20. In some embodiments, the degree of polymerization of the ethylene oxide grafted chlorinated polyethylene glycol is 3. In some embodiments, the degree of polymerization of the ethylene oxide grafted chlorinated polyethylene glycol is 20.

[0018] In some embodiments, the average molecular weight Mw of the chitosan is 4000-5000, for example, 4000, 4200, 4400, 4600, 4800, 5000 or any value therebetween.

[0019] In some embodiments, the reaction is carried out in a solvent, and the solvent includes an amide solvent, for example, N,N-dimethylformamide.

[0020] In some embodiments, the reaction is carried out in the presence of a first catalyst, which includes but is not limited to one or more of an alkali metal hydroxide and an alkali metal carbonate. In some embodiments, the first catalyst includes one or more of sodium carbonate, cesium carbonate, potassium carbonate, sodium hydroxide, and potassium hydroxide.

[0021] In some embodiments, the molar ratio of the first catalyst to the epoxidation reagent is 1:(1-3), for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3 or any value therebetween.

[0022] In some embodiments, the molar ratio of the chitosan to the epoxidation reagent is 1:(0.1-1.0), for example, 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 or any value therebetween.

[0023] In some embodiments, the reaction temperature is 0-95°C, preferably 15-40°C.

[0024] In some embodiments, the reaction time is 0.5-30 hours, preferably 12-24 hours.

[0025] In some embodiments, the molecular weight of the first sodium hyaluronate is 800-1500 KDa, for example, 800 Da, 1 KDa, 10 KDa, 30 KDa, 50 KDa, 80 KDa, 100 KDa, 200 KDa, 300 KDa, 400 KDa, 500 KDa, 800 KDa, 1000 KDa, 1100 KDa, 1200 KDa, 1300 KDa, 1400 KDa, 1500 KDa, or any value therebetween. In some embodiments, the molecular weight of the first sodium hyaluronate is 100 KDa-1000 KDa. In some embodiments, the molecular weight of the first sodium hyaluronate is 200 KDa-400 KDa.

[0026] In the present invention, the ethylene oxide groups grafted onto the modified chitosan are ring-opened and then cross-linked with the hydroxyl groups of the first sodium hyaluronate.

[0027] In some embodiments, the molar ratio of the first sodium hyaluronate to the modified chitin is 1:(0.1-1), for example, 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 or any value therebetween.

[0028] In some embodiments, the method for preparing cross-linked sodium hyaluronate comprises the following steps:

[0029] The aqueous solution of the modified chitosan is mixed with the aqueous solution of the first sodium hyaluronate and reacted to obtain the cross-linked sodium hyaluronate.

[0030] The modified chitosan grafted ethylene oxide group is ring-opened and cross-linked with the hydroxyl group of the first sodium hyaluronate. After cross-linking the first sodium hyaluronate, the modified chitosan can serve as a viscosity modifier to significantly reduce the viscosity of the first sodium hyaluronate.

[0031] In some embodiments, each 100 mL of the aqueous solution of the modified chitosan contains 0.5 to 2 g, preferably 0.8 to 1.2 g, of the modified chitosan.

[0032] In some embodiments, each 100 mL of the aqueous solution of the first sodium hyaluronate contains 0.5 to 2 g, preferably 0.8 to 1.2 g, of the first sodium hyaluronate.

[0033] In some embodiments, the volume ratio of the aqueous solution of modified chitosan to the aqueous solution of the first sodium hyaluronate is 1:(8-12), for example, 1:8, 1:9, 1:10, 1:11, 1:12 or any value therebetween.

[0034] In some embodiments, the reaction is performed at a pH of 8.0-9.0.

[0035] In some embodiments, the reaction temperature is 0-80°C, for example, 0°C, 10°C, 20°C, 25°C, 27°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C or any value therebetween. In some embodiments, the reaction temperature is 25-40°C.

[0036] In some embodiments, the reaction time is 0.5-30 hours, for example, 0.5 hours, 5 hours, 10 hours, 12 hours, 15 hours, 17 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours or any value therebetween. In some embodiments, the reaction time is 12-24 hours.

[0037] In some embodiments, the pH is adjusted to 6.5-7.0 after the reaction.

[0038] In some embodiments, the molecular weight of the second sodium hyaluronate is 800Da-1500KDa, for example, 800Da, 1KDa, 10KDa, 30KDa, 50KDa, 80KDa, 100KDa, 200KDa, 300KDa, 400KDa, 500KDa, 800KDa, 1000KDa, 1100KDa, 1200KDa, 1300KDa, 1400KDa, 1500KDa or any value therebetween. In some embodiments, the molecular weight of the second sodium hyaluronate is 100KDa-1500KDa. In some embodiments, the molecular weight of the second sodium hyaluronate is 300KDa-1300KDa. In some embodiments, the molecular weight of the second sodium hyaluronate is 1000KDa-1300KDa.

[0039] In some embodiments, the amount of the cross-linked sodium hyaluronate is 5-15% of the mass of the second sodium hyaluronate, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or any value therebetween. In some embodiments, the amount of the cross-linked sodium hyaluronate is 8-12% of the mass of the second sodium hyaluronate.

[0040] In some embodiments, the preparation method of the nano-cross-linked sodium hyaluronate comprises the following steps:

[0041] The aqueous solution of the cross-linked sodium hyaluronate and the aqueous solution of the second sodium hyaluronate are mixed and reacted to obtain the nano-cross-linked sodium hyaluronate.

[0042] According to some preferred embodiments of the present invention, the cross-linked sodium hyaluronate is used as a cross-linking agent and cross-linked with a second sodium hyaluronate again, which can significantly improve the viscosity and transdermal effect of the second sodium hyaluronate, effectively increase the water content of the skin, thereby hydrating the stratum corneum of the skin, and thereby increasing the permeability of the skin.

[0043] In some embodiments, each 100 mL of the aqueous solution of the cross-linked sodium hyaluronate contains 0.5 to 2 g, preferably 0.8 to 1.2 g, of the cross-linked sodium hyaluronate.

[0044] In some embodiments, each 100 mL of the aqueous solution of the second sodium hyaluronate contains 0.5 to 2 g, preferably 0.8 to 1.2 g, of the second sodium hyaluronate.

[0045] In some embodiments, the volume ratio of the aqueous solution of cross-linked sodium hyaluronate to the aqueous solution of the second sodium hyaluronate is 1:(5-15), for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, or any value therebetween, preferably 1:(8-12). In some embodiments, the volume ratio of the aqueous solution of cross-linked sodium hyaluronate to the aqueous solution of the second sodium hyaluronate is 1:10.

[0046] In some embodiments, the mass ratio of the cross-linked sodium hyaluronate to the second sodium hyaluronate is 1:(0.01-1000), for example, 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1.0, 1:1.5, 1:2, 1:5, 1:10, 1:20, 1:50, 1:80, 1:100, 1:200, 1:500, 1:800, 1:1000, or any value therebetween. In some embodiments, the mass ratio of the cross-linked sodium hyaluronate to the second sodium hyaluronate is 1:(0.1-100). In some embodiments, the mass ratio of the cross-linked sodium hyaluronate to the second sodium hyaluronate is 1:(0.1-10). In some embodiments, the mass ratio of the cross-linked sodium hyaluronate to the second sodium hyaluronate is 1:(1-10). In some embodiments, the mass ratio of the cross-linked sodium hyaluronate to the second sodium hyaluronate is 1:(0.1-1).

[0047] In some embodiments, the reaction temperature is 0-80°C, preferably 25-40°C.

[0048] In some embodiments, the reaction time is 0.5-30 hours, preferably 12-24 hours.

[0049] According to some preferred embodiments of the present invention, nano-cross-linked sodium hyaluronate is prepared by mixing the aforementioned cross-linked sodium hyaluronate with sodium hyaluronate. By adjusting the molecular weight of the sodium hyaluronate and the ratio of cross-linked sodium hyaluronate added, the size of the nano-cross-linked sodium hyaluronate particles is adjusted, thereby varying the amount of water absorbed by the skin surface, thereby improving the efficiency of the nano-cross-linked sodium hyaluronate system in promoting the penetration of active ingredients into the skin.

[0050] In a second aspect, the present invention provides a method for preparing the antioxidant composition described in the first aspect, comprising the following steps: dissolving nano-cross-linked sodium hyaluronate in a solvent to obtain solution A; and mixing an antioxidant with solution A.

[0051] According to an embodiment of the present invention, the solvent is water.

[0052] According to an embodiment of the present invention, the mass concentration of nano-cross-linked sodium hyaluronate in solution A is 0.001%-50%, for example, 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%.

[0053] According to an embodiment of the present invention, the mass concentration of the antioxidant in the solution A is 0.001%-50%, 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%.

[0054] According to an embodiment of the present invention, the preparation method of the antioxidant composition includes the following steps: dissolving nano-cross-linked sodium hyaluronate in a solvent to obtain solution A; dissolving an antioxidant in a solvent to obtain solution B; and mixing solution A and solution B.

[0055] According to an embodiment of the present invention, the mass concentration of the antioxidant in the solution B is 0.001%-50%, 0.001%, 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%, 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%.

[0056] According to an embodiment of the present invention, the mixing conditions include: a stirring speed of 10-2000 rpm and a stirring time of 0.1-48 h.

[0057] In a third aspect, the present invention further provides a skin care product comprising the antioxidant composition described in the first aspect of the present invention or the antioxidant composition prepared by the preparation method described in the second aspect of the present invention.

[0058] In some embodiments, the skin care products include but are not limited to skin care water, skin care lotion, skin care cream, skin care essence, skin care mask, etc.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The antioxidant composition provided by the present invention utilizes the specific encapsulation effect of nano-sodium hyaluronate to improve the transdermal absorption efficiency of the antioxidant, and has broad application prospects in the field of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 IR spectra of the products obtained in Example 2 and Example 3B.

[0062] Figure 2 4B and sodium hyaluronate (300,000 Da).

[0063] Figure 3 The total reflection infrared spectra of the mouse epidermis were treated with the products of Example 3A, Example 3B, Example 3C, and the product of Example 4B cross-linked with 1.3 million Da molecular weight sodium hyaluronate.

[0064] Figure 4 The epidermal water content is measured after the epidermis is treated with the products of Example 3A, Example 3B, and Example 4A, and a 1,000,000 Da molecular weight sodium hyaluronate solution.

[0065] Figure 5 This is the fluorescence imaging image of the transdermal experiment in Experiment 3.

[0066] Figure 6 yes Figure 5 Statistical graph of fluorescence imaging results. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, 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 illustrate the present invention and are not intended to constitute any limitation to the present invention.

[0068] Unless otherwise specified, the reagents used in the following experiments were commercially available or prepared according to conventional methods. The methods used in the experiments were conventional experimental methods unless otherwise specified. The instruments used in the experiments were commercially available unless otherwise specified.

[0069] The room temperature in the present invention refers to 15-30°C.

[0070] Unless otherwise specified, "%" in the present invention refers to weight percentage.

[0071] Example 1 Synthesis of Propylene Oxide Grafted Chitosan

[0072] 5 g of chitosan (average molecular weight Mw ~4400) was dissolved in N,N-dimethylformamide solution, 0.5 g of sodium carbonate was added, and 1 g of epichlorohydrin was added after stirring. The reaction was allowed to react at room temperature for 24 hours. 50 mL of water was then added to quench the reaction. The solution was dialyzed using a 500 Da dialysis membrane to obtain an ethylene oxide-grafted chitosan solution. Propylene oxide-grafted chitosan was then freeze-dried to obtain the product.

[0073] Example 2 Synthesis of ethylene oxide polyethylene glycol grafted chitosan

[0074] 5 grams of chitosan (average molecular weight Mw ~ 4400) was dissolved in N,N-dimethylformamide solution, 0.5 grams of cesium carbonate was added, and after stirring, 1 gram of ethylene oxide-grafted chlorinated polyethylene glycol (DP = 20, CAS: 155101-67-0) was added. The mixture was allowed to react at room temperature for 24 hours. 50 mL of water was then added to quench the reaction. The solution was dialyzed using a 500 Da dialysis membrane to obtain an ethylene oxide-grafted chitosan solution. Freeze-dried, the product, ethylene oxide-grafted chitosan, was obtained. Analysis by a Thermo FlashSmart elemental analyzer revealed the following elemental weight percentages: N: 4.65%, C: 43.73%, H: 7.39%, and O: 44.23%. The infrared spectrum of the product is shown in Figure 2. Figure 1 As shown, it can be seen that at 1000-1200cm -1 There is a significant epoxy group vibration peak on the main vibration peak of COC on polyethylene glycol, indicating that the epoxy group has been connected to the surface of PEGylated chitosan. At the same time, at 1650-1580cm -1 The in-plane bending vibration absorption peak of primary amine is shown at .

[0075] Example 3A Synthesis of Propylene Oxide Grafted Chitosan Cross-linked Sodium Hyaluronate (1 Million Da)

[0076] Step 1: Take 0.1 g of propylene oxide grafted chitosan prepared in Example 1, add 10 mL of water, and stir to dissolve;

[0077] Step 2: Take 1g of sodium hyaluronate (1 million Da) and dissolve it in 100mL of water.

[0078] Step three, the solution prepared in step one is added to the solution prepared in step two, the pH is adjusted to 8-9 using 10% NaOH solution, then stirred at room temperature for 24 hours, and then the pH is adjusted to 6.5-7 using 10% HC1 solution.

[0079] Step four, the liquid obtained in step three is dialyzed through a dialysis membrane with MW 10000 Da, and then freeze-dried to obtain the product of propylene oxide grafted chitosan cross-linked sodium hyaluronate (100 million Da).

[0080] Example 3B Synthesis of propylene oxide grafted chitosan cross-linked sodium hyaluronate (10 million Da)

[0081] The difference from Example 3A is that "sodium hyaluronate (100 million Da)" in step two is replaced by "sodium hyaluronate (10 million Da)".

[0082] The final product, propylene oxide grafted chitosan cross-linked sodium hyaluronate (10 million Da), is analyzed by Thermo Flash Smart elemental analyzer, and the mass percentage of each element is: N: 4.79%, C: 46.05%, H: 7.51%, O: 41.65%. The infrared spectrum of the product is shown in Figure 1 Compared with Example 2, Example 3B has a significant increase in the proportion of stretching vibration peaks at 1000-1200 cm -1 The infrared spectrum of the product is shown in

[0083] Example 3C Synthesis of propylene oxide grafted chitosan cross-linked sodium hyaluronate (30 million Da)

[0084] The difference from Example 3A is that "sodium hyaluronate (100 million Da)" in step two is replaced by "sodium hyaluronate (30 million Da)".

[0085] Example 4A Synthesis of ethylene oxide polyethylene glycol grafted chitosan cross-linked sodium hyaluronate (10 million Da)

[0086] Step one, 0.1 g of ethylene oxide polyethylene glycol grafted chitosan prepared in Example 2 is added to 10 mL of water and stirred to dissolve;

[0087] Step two, 1 g of sodium hyaluronate (10 million Da) is dissolved in 100 mL of water.

[0088] Step 3: Add the solution prepared in step 1 to the solution prepared in step 2, adjust the pH to 8-9 with 10% NaOH solution, then stir at room temperature for 24 hours, and then adjust the pH to 6.5-7 with 10% HCl solution.

[0089] Step 4: The liquid obtained in step 3 is dialyzed through a dialysis membrane with a MW of 10,000 Da, and then freeze-dried to obtain the finished product of ethylene oxide polyethylene glycol-grafted chitosan cross-linked sodium hyaluronate (100,000 Da).

[0090] Example 4B Synthesis of Ethylene Oxide Polyethylene Glycol Grafted Chitosan Cross-linked Sodium Hyaluronate (300,000 Da)

[0091] Step 1: Take 0.1 g of ethylene oxide polyethylene glycol grafted chitosan prepared in Example 2, add 10 mL of water, and stir to dissolve;

[0092] Step 2: Take 1g of sodium hyaluronate (300,000 Da) and dissolve it in 100mL of water.

[0093] Step 3: Add the solution prepared in step 1 to the solution prepared in step 2, adjust the pH to 8-9 with 10% NaOH solution, then stir at room temperature for 24 hours, and then adjust the pH to 6.5-7 with 10% HCl solution.

[0094] Step 4: The liquid obtained in step 3 was dialyzed through a dialysis membrane with a MW of 10,000 Da and then freeze-dried to obtain the finished product, ethylene oxide polyethylene glycol grafted chitosan cross-linked sodium hyaluronate (300,000 Da). Analysis by a Thermo FlashSmart elemental analyzer revealed the weight percentages of the elements in the finished product to be 3.22% N, 36.66% C, and 5.36% H. The infrared spectra of sodium hyaluronate (300,000 Da) and the product, ethylene oxide polyethylene glycol grafted chitosan cross-linked sodium hyaluronate (300,000 Da), are shown in Figure 2. Figure 2 shown.

[0095] Example 4C Synthesis of Ethylene Oxide Polyethylene Glycol Grafted Chitosan Cross-linked Sodium Hyaluronate (1 Million Da)

[0096] Step 1: Take 0.1 g of ethylene oxide polyethylene glycol grafted chitosan prepared in Example 2, add 10 mL of water, and stir to dissolve;

[0097] Step 2: Take 1g of sodium hyaluronate (1 million Da) and dissolve it in 100mL of water.

[0098] Step 3: Add the solution prepared in step 1 to the solution prepared in step 2, adjust the pH to 8-9 with 10% NaOH solution, then stir at room temperature for 24 hours, and then adjust the pH to 6.5-7 with 10% HCl solution.

[0099] Step 4: The liquid obtained in step 3 is dialyzed through a dialysis membrane with a MW of 10,000 Da and then freeze-dried to obtain the finished product of ethylene oxide polyethylene glycol-grafted chitosan cross-linked sodium hyaluronate (1 million Da).

[0100] Example 5 Synthesis of Ethylene Oxide Polyethylene Glycol Grafted Chitosan

[0101] The difference from Example 2 is that "ethylene oxide grafted chlorinated polyethylene glycol (polymerization degree = 20, CAS: 155101-67-0)" is replaced by "ethylene oxide grafted chlorinated polyethylene glycol (polymerization degree = 3, CAS: 155101-68-1)".

[0102] Example 6 Synthesis of Ethylene Oxide Polyethylene Glycol Grafted Chitosan Cross-linked Sodium Hyaluronate (300,000 Da)

[0103] The only difference from Example 4B is that in step 1, the "ethylene oxide polyethylene glycol grafted deacetylated chitosan prepared in Example 2" is replaced with "ethylene oxide polyethylene glycol grafted deacetylated chitosan prepared in Example 5" to obtain ethylene oxide polyethylene glycol grafted deacetylated chitosan cross-linked sodium hyaluronate (300,000 Da).

[0104] Comparative Example 1 Synthesis of Hydroxyethyl Chitosan Cross-linked Sodium Hyaluronate

[0105] Dissolve 1 g of sodium hyaluronate (200,000-400,000 Da) in 50 mL of water, then add 3 g of the prepared hydroxyethyl chitosan (average molecular weight Mw ~ 8000). After dissolution, add 0.1 mL of glutaraldehyde and react at room temperature for 12 hours. The reactant is dialyzed and freeze-dried to prepare 1.5 g of the product hydroxyethyl chitosan cross-linked sodium hyaluronate (200,000-400,000 Da).

[0106] Comparative Example 2 Preparation of cross-linked sodium hyaluronate using carboxymethyl chitosan as a cross-linking agent

[0107] To 30 mL of N,N-dimethylformamide, 5 g of carboxymethyl chitosan (average molecular weight Mw ~ 160,000) was added, stirred evenly, and then 0.1 mL of thionyl chloride was added. The mixture was reacted at 90°C for 8 hours. Then, 5 g of sodium hyaluronate (1 million Da) in 50 mL of N,N-dimethylformamide was added. After the reaction was continued for 6 hours, 300 mL of acetone was added to precipitate. After filtering to remove the solvent, the precipitate was washed with 3×30 mL of acetone to obtain carboxymethyl chitosan cross-linked sodium hyaluronate (1 million Da).

[0108] Example 7 Synthesis of Nano-cross-linked Sodium Hyaluronate

[0109] Step 1: Take 0.1 g of propylene oxide-grafted chitosan cross-linked sodium hyaluronate (1,000,000 Da) prepared in Example 3A as a cross-linking agent, add 10 mL of water and stir to dissolve;

[0110] Step 2: Take 1 g of sodium hyaluronate (1.3 million Da) and dissolve it in 100 mL of water to obtain a sodium hyaluronate solution;

[0111] Step 3: Add the solution prepared in step 1 to the sodium hyaluronate solution prepared in step 2, and stir at room temperature for 48 hours to obtain a nano-cross-linked sodium hyaluronate solution;

[0112] Step 4: The nano-cross-linked sodium hyaluronate solution obtained in step 3 is dialyzed through a dialysis membrane with a MW of 10,000 Da and then freeze-dried to obtain the finished product - nano-cross-linked sodium hyaluronate.

[0113] Example 8-Example 16 Synthesis of Nano-cross-linked Sodium Hyaluronate

[0114] The same method as in Example 7 was used to adjust the cross-linking agent used in step 1 and the molecular weight of sodium hyaluronate used in step 2 according to the formula in Table 1 below to obtain different nano-cross-linked sodium hyaluronates.

[0115] Table 1

[0116]

[0117]

[0118] The average particle size of the nano-cross-linked sodium hyaluronate products in each example in Table 1 was measured using a laser particle size analyzer, a Zetasizer Nano ZS manufactured by Malvern Instruments Ltd., UK, with a measurement angle θ of 173°; the sample cell temperature was adjustable within the range of 2-90°C; and the laser wavelength used was 633 nm.

[0119] Experiment 1: Viscosity Test

[0120] The propylene oxide / ethylene oxide polyethylene glycol-grafted chitosan-crosslinked sodium hyaluronate prepared in Examples 1, 2, 3A, 3B, 3C, 4A, 4B, 4C, 5, 6 and Comparative Examples 1-2 was used as a crosslinking agent and mixed with sodium hyaluronate solutions of different molecular weights to obtain nano-crosslinked sodium hyaluronate solutions. The viscosity change of the nano-crosslinked sodium hyaluronate solutions was examined.

[0121] In the following experiments, the bulk viscosity of sodium hyaluronate solution was measured using an NDJ-8S digital display viscometer (Shanghai Precision Scientific Instrument Co., Ltd.) at a constant temperature of 298K. The test method is as follows:

[0122] (1) Preparation of sodium hyaluronate solutions with different molecular weights: Dissolve 1 g of sodium hyaluronate with a molecular weight of 1.3 million Da, 1 g of sodium hyaluronate with a molecular weight of 1 million Da, and 1 g of sodium hyaluronate with a molecular weight of 200,000 Da to 400,000 Da in 100 mL of water to prepare 1% sodium hyaluronate (1.3 million Da) solution, 1% sodium hyaluronate (1 million Da) solution, and 1% sodium hyaluronate (200,000 Da to 400,000 Da) solution, respectively;

[0123] (2) Preparation of crosslinker solution: Dissolve 1 g of crosslinker in 100 mL of water to prepare a 1% crosslinker solution.

[0124] (3) Weigh 10 mL of 1% cross-linking agent solution and add it to 90 mL of the 1% sodium hyaluronate solution prepared in step (1). After standing for 0.5-48 hours, a nano-cross-linked sodium hyaluronate solution is obtained.

[0125] The viscosity of a 1% sodium hyaluronate (1.3 million Da) solution was tested to be 13,674 mPa / S;

[0126] The viscosity of a 1% sodium hyaluronate (1 million Da) solution is 12109 mPa / S;

[0127] The viscosity of a 1% sodium hyaluronate (200,000 Da-400,000 Da) solution is 6832 mPa / S;

[0128] When the ethylene oxide polyethylene glycol grafted chitosan cross-linked sodium hyaluronate (300,000 Da) prepared in Example 4B was prepared into a 1% concentration cross-linking agent solution, the viscosity of the cross-linking agent solution was 126 mPa / S.

[0129] Some experimental results are shown in Table 2.

[0130] Table 2

[0131]

[0132]

[0133] It can be seen from Table 2 above that the cross-linked sodium hyaluronate prepared in Examples 3A-3C, 4A-4C, and Example 6 of the present application can significantly reduce the viscosity of the sodium hyaluronate solution as a cross-linking agent; while the sodium hyaluronate cross-linked with hydroxyethyl chitosan prepared in Comparative Example 1 and the sodium hyaluronate cross-linked with carboxymethyl chitosan prepared in Comparative Example 2 cannot change the viscosity and state of the sodium hyaluronate.

[0134] Experiment 2: Skin epidermis experiment

[0135] (I) ATR-FTIR analysis of mouse epidermal (SC) samples

[0136] Step 1: Preparation of mouse epidermis

[0137] Kunming mice were sacrificed and their hair removed with electric clippers. The skin was then carefully shaved with a razor and obtained. The epidermis was gently peeled off by trimming the subcutaneous fat and immersing the full-thickness skin sample in water. Stratum corneum sheets were obtained by floating the freshly prepared epidermis in an aqueous solution containing 0.0001% trypsin and 0.5% sodium bicarbonate for 24 hours. The digest beneath the stratum corneum was removed with filter paper, and the separated sheets were rinsed in acetone for 30 seconds to remove any sebum or subcutaneous fat contamination, then freeze-dried and stored in silica gel under vacuum.

[0138] Step 2: Preparation of different sodium hyaluronate samples

[0139] Sodium hyaluronate sample 1: Dissolve 1 g of 1.3 million Da sodium hyaluronate in 100 mL of water to prepare a 1% sodium hyaluronate (1.3 million Da) solution.

[0140] Sodium Hyaluronate Sample 2: 1 g of the product prepared in Example 4B was dissolved in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (300,000 Da) solution; 10 mL of the 1% cross-linked sodium hyaluronate (300,000 Da) solution was added to 90 mL of a 1% sodium hyaluronate (1.3 million Da) solution (prepared as in Sodium Hyaluronate Sample 1). After standing for 0.5-48 hours, a nano-cross-linked sodium hyaluronate solution was obtained, recorded as 1% sodium hyaluronate (1.3 million Da) + 1% cross-linked sodium hyaluronate (300,000 Da).

[0141] Sodium hyaluronate sample 3: 1 g of the product prepared in Example 3A was dissolved in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate solution (1 million Da).

[0142] Sodium hyaluronate sample 4: 1 g of the product prepared in Example 3B was dissolved in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate solution (100,000 Da);

[0143] Sodium hyaluronate sample 5: 1 g of the product prepared in Example 3C was dissolved in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (300,000 Da) solution.

[0144] Step 3: Testing

[0145] A 6 mm × 6 mm dry SC sample was incubated in 3 ml of sodium hyaluronate samples 1 to 5 prepared in step 2 above and a blank solvent (water, as a control) at room temperature for 24 hours. Subsequently, the SC piece was carefully washed with distilled water to remove all residual solvents on its surface. After freeze-drying, the spectra were measured using a Nicolet FTIR 5700 Fourier transform infrared spectrometer equipped with an ATR accessory (Thermo Fisher Scientific, Inc., USA) under the following conditions: single crystal diamond crystal mirror, scanning temperature range of 18–20 degrees Celsius, number of scans of 64 times, resolution of 4 cm^-1, and scanning range of 800–4000 cm^-1. Infrared spectra as shown Figure 3 Each set of curves was recorded and analyzed using second-order derivatives, deconvolution, and curve fitting. The specific method is as follows: Peakfit v4.12 software was used to analyze the curves under the following conditions. Figure 4 Fourier self-deconvolution and curve fitting were performed with a tolerance of 9%, a deconvolution width of 3.00, a filter of 55.0, and peak types of spectroscopy and Gaussian area. The height, width, and position of each peak were optimized sequentially. The relative areas of the amide I region peaks were calculated based on the final fitted peak areas. The stratum corneum secondary structure content was determined based on the peak areas. The results are shown in Table 3.

[0146] Table 3 Changes in the secondary structure of the skin stratum corneum in different treatment groups

[0147]

[0148] As can be seen from Table 3, the cross-linked sodium hyaluronate prepared in Example 4B can more significantly improve the β-folding of the stratum corneum and promote the skin absorption of the efficacy substance after re-cross-linking with sodium hyaluronate (130W).

[0149] (II): ATR-FTIR analysis of water content in mouse epidermis (SC)

[0150] Step 1: Preparation of mouse epidermis

[0151] Kunming mice were sacrificed and their hair removed with electric clippers. The skin was then carefully shaved with a razor and obtained. The epidermis was gently peeled off by trimming the subcutaneous fat and immersing the full-thickness skin sample in water. Stratum corneum sheets were obtained by floating the freshly prepared epidermis in an aqueous solution containing 0.0001% trypsin and 0.5% sodium bicarbonate for 24 hours. The digest beneath the stratum corneum was removed with filter paper, and the separated sheets were rinsed in acetone for 30 seconds to remove any sebum or subcutaneous fat contamination, then freeze-dried and stored in silica gel under vacuum.

[0152] Step 2: Preparation of different sodium hyaluronate samples

[0153] Sodium hyaluronate sample 1: Dissolve 1 g of 1 million Da sodium hyaluronate in 100 mL of water to prepare a 1% sodium hyaluronate (1 million Da) solution.

[0154] Sodium hyaluronate sample 2: 1 g of the product prepared in Example 3A was dissolved in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate solution (1 million Da).

[0155] Sodium hyaluronate sample 3: 1 g of the product prepared in Example 3B was dissolved in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate solution (100,000 Da);

[0156] Sodium hyaluronate sample 4: 1 g of the product prepared in Example 4A was dissolved in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (100,000 Da) solution.

[0157] Step 3: Testing

[0158] A 6 mm x 6 mm dry SC sample was placed in 3 ml of sodium hyaluronate samples 1 to 4 prepared in step 2 above and a blank solvent (water, as a control) and incubated at room temperature, and the operating steps were the same as described above. At predetermined time intervals (0, 2, 4, 6, 8, 12 hours), the SC sample was taken out and carefully washed with distilled water to remove the residual solvent on the surface. Subsequently, the residual moisture on the surface of the SC was absorbed with filter paper, and the moisture content in the SC was detected by ATR-FTIR under the above conditions. The moisture content in the SC was determined by calculating the ratio of the amide I absorption peak to the amide II absorption peak. The results are shown in FIG. Figure 4 As shown, compared with other examples, the chitosan cross-linked sodium hyaluronate grafted with ethylene oxide polyethylene glycol prepared in Example 4A can more significantly improve the water content of the stratum corneum and promote the skin absorption of the efficacy substance.

[0159] Experiment 3

[0160] 1. Test sample preparation

[0161] Take 0.1g Cy3-COOH (0.594g) and dissolve it in 10mL DMF. At 0℃, add 0.15g thionyl chloride. After stirring for half an hour, return to room temperature, evaporate the excess thionyl chloride, and redissolve it in 5mL DMF.

[0162] 3.84 g of bosine was weighed and dissolved in 20 mL of DMF. The Cy3-COOH acyl chloride prepared in the previous step was then added. The mixture was stirred at room temperature overnight. Fluorescently labeled bosine A was obtained by column chromatography (methanol: dichloromethane = 9.5:1).

[0163] 0.1 g of the prepared fluorescently labeled bosonin A was dissolved in 5 mL of an aqueous solution containing 0.5% hydroxyethyl chitosan cross-linked sodium hyaluronate to obtain a fluorescently labeled bosonin product B.

[0164] 0.1 g of the prepared fluorescently labeled hyaluronan A was dissolved in 5 mL of an aqueous solution containing 0.5% of the nano-cross-linked sodium hyaluronate prepared in Example 13 to obtain a fluorescently labeled hyaluronan product C.

[0165] 0.1 g of the prepared fluorescently labeled hyaluronan A was dissolved in 5 mL of an aqueous solution containing 0.5% of the nano-cross-linked sodium hyaluronate prepared in Example 8 to obtain a fluorescently labeled hyaluronan product D.

[0166] 0.1 g of the prepared fluorescently labeled hyaluronan A was dissolved in 5 mL of an aqueous solution containing 0.5% of the nano-cross-linked sodium hyaluronate prepared in Example 11 to obtain a fluorescently labeled hyaluronan product E.

[0167] 0.1 g of the prepared fluorescently labeled bosonin A was dissolved in 5 mL of an aqueous solution containing 0.5% carboxymethyl chitosan cross-linked sodium hyaluronate to obtain a fluorescently labeled bosonin product F.

[0168] 2. Transdermal test

[0169] Sixty C57BL / 6 mice were randomly divided into six groups, with 10 mice in each group, namely the control group, A, B, C, D, E, and F. Two 5*5 cm patches of hair were removed from each mouse using an electric shaver to expose the skin. Then, 0.5 mL of the prepared blank solution (ultrapure water, control) was applied to the two depilated skin patches, A, B, C, D, E, and F. After eight hours, the mice were anesthetized and sacrificed. The skin patches applied with the blank solution (ultrapure water, control) and A, B, C, D, E, and F were routinely prepared into paraffin sections for fluorescence imaging, as shown in Figure 2. Figure 5 shown.

[0170] 3. Fluorescence quantitative statistical method

[0171] SPSS 13.0 statistical software was used to analyze the data. The results of direct immunofluorescence staining of frozen sections were compared using the x2 test, with P < 0.05 considered statistically significant. Figure 6 It should be noted that Figure 5In the table, C-HA represents sodium hyaluronate cross-linked with hydroxymethyl chitosan; BOSE represents fluorescently labeled BOSE A; BOSE@CS / HA represents fluorescently labeled BOSE product B; BOSE@nano sodium hyaluronate (20-40W) represents fluorescently labeled BOSE product C; BOSE@nano sodium hyaluronate (100W) represents fluorescently labeled BOSE product D; BOSE@nano sodium hyaluronate (10W) represents fluorescently labeled BOSE product E; BOSE@C-HA represents fluorescently labeled BOSE product F.

[0172] 4. Results

[0173] from Figure 5 and Figure 6 It can be concluded that, compared with hyaluronan monomer, the nano-cross-linked sodium hyaluronate of the present invention can significantly promote the transdermal absorption of hyaluronan, and the absorption amount is more than 3 times that of other reference products.

[0174] Furthermore, compared with hydroxymethyl or hydroxyethyl chitosan cross-linked sodium hyaluronate, the absorption capacity of the nano-cross-linked sodium hyaluronate of the present invention is also significantly increased, among which the nano-cross-linked sodium hyaluronate with a molecular weight of 200,000-400,000 Da has a higher absorption capacity.

[0175] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An antioxidant composition comprising nano-cross-linked sodium hyaluronate and an antioxidant, The nano-cross-linked sodium hyaluronate includes a second sodium hyaluronate cross-linked by cross-linked sodium hyaluronate, and the cross-linked sodium hyaluronate includes a first sodium hyaluronate cross-linked by modified chitin; in, The modified chitosan comprises a chitosan backbone, and the backbone is grafted via ether bonds. In the group, p is an integer between 0 and 200, preferably an integer between 1 and 200, more preferably an integer between 1 and 20, and further preferably an integer between 3 and 20.

2. The antioxidant composition according to claim 1, characterized in that p is an integer between 1 and 200; and / or The weight ratio of the nano-cross-linked sodium hyaluronate to the antioxidant is 1:(1-50), and / or The antioxidant is selected from one or more of ergothioneine, bosine or ectoine; and / or The nano-cross-linked sodium hyaluronate and the antioxidant form nanoparticles.

3. The antioxidant composition according to claim 1, characterized in that The preparation method of the modified chitosan comprises the following steps: reacting deacetylated chitosan with an epoxidation agent to obtain the modified chitosan; wherein the epoxidation agent comprises epichlorohydrin and / or chlorinated polyethylene glycol grafted with ethylene oxide; Preferably, the degree of polymerization of the ethylene oxide grafted chlorinated polyethylene glycol is 1 to 200, preferably 1 to 100, more preferably 1 to 20, and further preferably 3 to 20; Preferably, the reaction is carried out in a solvent, and the solvent includes an amide solvent; Preferably, the reaction is carried out in the presence of a first catalyst, preferably, the first catalyst comprises one or more of an alkali metal hydroxide and an alkali metal carbonate; Preferably, the molar ratio of the first catalyst to the epoxidation reagent is 1:(1-3); Preferably, the molar ratio of the chitosan to the epoxidation reagent is 1:(0.1-1.0); Preferably, the reaction temperature is 0-95°C, preferably 15-40°C; Preferably, the reaction time is 0.5-30 hours, preferably 12-24 hours; Preferably, the average molecular weight of the chitosan is 4000-5000.

4. The antioxidant composition according to claim 1, characterized in that The molecular weight of the first sodium hyaluronate is 800Da-1500KDa, preferably 100KDa-1000KDa, more preferably 200KDa-400KDa; The molar ratio of the first sodium hyaluronate to the modified chitin is 1:(0.1-1).

5. The antioxidant composition according to claim 1, characterized in that The preparation method of the cross-linked sodium hyaluronate comprises the following steps: The aqueous solution of the modified chitosan is mixed with the aqueous solution of the first sodium hyaluronate and reacted to obtain the cross-linked sodium hyaluronate.

6. The antioxidant composition according to claim 5, characterized in that Each 100 mL of the aqueous solution of the modified chitosan contains 0.5 to 2 g of the modified chitosan; Each 100 mL of the aqueous solution of the first sodium hyaluronate contains 0.5 to 2 g of the first sodium hyaluronate; The volume ratio of the aqueous solution of modified chitosan to the aqueous solution of the first sodium hyaluronate is 1:(8-12); The reaction is carried out at a pH of 8.0-9.0; The reaction temperature is 0-80°C; The reaction time is 0.5-30 hours; After the reaction, the pH was adjusted to 6.5-7.

0.

7. The antioxidant composition according to any one of claims 1 to 6, characterized in that The molecular weight of the second sodium hyaluronate is 800Da-1500KDa, preferably 100KDa-1500KDa, more preferably 1000KDa-1300KDa; and / or The amount of the cross-linked sodium hyaluronate is 5-15% of the mass of the second sodium hyaluronate, preferably 8-12%.

8. The antioxidant composition according to any one of claims 1 to 6, characterized in that The preparation method of the nano-cross-linked sodium hyaluronate comprises the following steps: Mixing the aqueous solution of the cross-linked sodium hyaluronate and the aqueous solution of the second sodium hyaluronate and reacting them to obtain the nano-cross-linked sodium hyaluronate; Preferably, each 100 mL of the aqueous solution of the cross-linked sodium hyaluronate contains 0.5 to 2 g of the cross-linked sodium hyaluronate; Preferably, each 100 mL of the aqueous solution of the second sodium hyaluronate contains 0.5 to 2 g of the second sodium hyaluronate; Preferably, the volume ratio of the aqueous solution of cross-linked sodium hyaluronate to the aqueous solution of the second sodium hyaluronate is 1:(5-15); Preferably, the reaction temperature is 0-80°C; Preferably, the reaction time is 0.5-30 hours.

9. A method for preparing the antioxidant composition according to any one of claims 1 to 8, comprising the steps of: dissolving nano-cross-linked sodium hyaluronate in a solvent to obtain solution A; mixing an antioxidant with solution A; The solvent is water; The mass concentration of nano-cross-linked sodium hyaluronate in the solution A is 0.001%-50%. The mass concentration of the antioxidant in the solution A is 0.001%-50%; The mixing conditions include: The stirring speed is 10-2000 rpm, and / or the stirring time is 0.1-48 h.

10. A skin care product comprising the antioxidant composition according to any one of claims 1 to 8 or the antioxidant compound prepared by the preparation method according to claim 9; The skin care product includes any one of skin care lotion, skin care lotion, skin care cream, skin care essence, and skin care mask.

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