Antioxidant composition as well as preparation method and application thereof

Abstract

The invention discloses an antioxidant composition as well as a preparation method and application thereof. The antioxidant composition comprises a composite sulfhydrylation hyaluronic acid solution, a leontopodium alpinum extract and a scutellaria alpina extract, the composite sulfhydrylated hyaluronic acid solution comprises sulfhydrylated hyaluronic acid oligosaccharide with the molecular weight of 1.2 kDa to 10 kDa and low-molecular-weight sulfhydrylated hyaluronic acid with the molecular weight of 100 kDa to 300 kDa, the sulfydryl content of the sulfhydrylated hyaluronic acid oligosaccharide and the sulfydryl content of the low-molecular-weight sulfhydrylated hyaluronic acid are both 37.5 mu mol / g to 375 mu mol / g, and the total content of hyaluronic acid in the composite sulfhydrylated hyaluronic acid solution is 0.01 mg / mL to 30 mg / mL. The preparation method of the anti-oxidation composition is simple, the three components are organically combined, the anti-oxidation and barrier repairing effects are achieved, and the anti-oxidation composition is especially suitable for crowds with sensitive skin and suitable for being applied to preparation of anti-oxidation cosmetics.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an antioxidant composition and a preparation method and application thereof. Background Art

[0002] In the normal metabolism of the human body, there is a dynamic balance between oxidation and reduction. However, under the influence of various endogenous factors (such as chronic or acute infections) and exogenous factors (such as environmental pollution, ultraviolet radiation, etc.), oxidation reactions may take the lead, resulting in oxidative stress, which in turn produces a variety of harmful reactive oxygen free radicals; reactive oxygen free radicals are highly oxidizing and will attack lipids, proteins, and DNA, etc., which can damage the body's tissues and cells, and then cause various chronic diseases and aging effects such as dryness, roughness, dullness, sagging, and wrinkles.

[0003] The skin barrier of sensitive people is more easily damaged, and their ability to resist the harsh external environment is weaker. Seasonal changes, staying up late, air pollution and ultraviolet rays may all induce sensitive skin. At this time, sensitive skin produces more harmful free radicals, making it more likely to experience oxidative stress and cause skin aging.

[0004] Hyaluronic acid, also known as hyaluronic acid, is an acidic mucopolysaccharide inherent in the human body. It is widely used in various skin care products. It is a super moisturizer naturally present in human skin and can maintain 1000 times its own weight of water. The depth of transdermal absorption into the skin is related to the molecular weight of hyaluronic acid. Among them, oligosaccharides with a molecular weight of less than 10KDa can penetrate into the deep dermis more than 100 microns from the skin surface, and low molecular weight hyaluronic acid with a molecular weight of 100kDa to 300kDa can penetrate to the epidermis about 50 microns from the skin surface (M.Essendoubi et al., Skin Research and Technology 2016, 22: 55-62). Hyaluronic acid that enters the skin can remove active oxygen free radicals. However, the ability of such quenching active oxygen free radicals is very weak and insufficient to effectively remove active oxygen free radicals (Yamazaki et al., Pathophysiology 2003, 9: 215-220; Stern et al., Biotechnology Advances 2007, 25: 537-557; Serban et al., Biomaterials 2008, 29: 1388-1399).

[0005] Thiol is a highly active group that can effectively inhibit oxidative stress directly caused by reactive oxygen free radicals and prevent damage to lipids, proteins and DNA. In addition, it can also indirectly inhibit the formation of hydroxyl free radicals by complexing with divalent iron ions. Therefore, thiol modification of hyaluronic acid is an effective way to improve the ability to scavenge reactive oxygen free radicals.

[0006] The antioxidant strategy for people with sensitive skin is to both remove harmful free radicals and repair the skin barrier. The combination of the two can synergistically exert a three-dimensional antioxidant effect. Although there are some technical examples of skin antioxidant, the antioxidant strategy of the prior art does not have both of the above two points. Therefore, it is necessary to develop a composition that combines thiolated hyaluronic acid with other substances with antioxidant and barrier repair effects, and has the activity enhancement effect of removing harmful free radicals at different depths of the skin and repairing the skin barrier. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an antioxidant composition and a preparation method and application thereof, wherein the antioxidant composition not only has the function of eliminating harmful free radicals in the entire skin layer and repairing the skin barrier, but also the synergistic effect of the composite thiolated hyaluronic acid, alpine edelweiss and alpine scutellaria further makes it have a comprehensive antioxidant effect with obvious effect and long duration, and is especially suitable for people with sensitive skin and suitable for application in the field of cosmetics; and the preparation method of the antioxidant composition of the present invention is simple.

[0008] In order to solve the above technical problems, the first aspect of the present invention is to provide an antioxidant composition, comprising the following components:

[0009] Complex thiolated hyaluronic acid solution;

[0010] Alpine edelweiss extract;

[0011] Scutellaria alpinia extract;

[0012] The composite thiolated hyaluronic acid solution comprises thiolated hyaluronic acid oligosaccharides with a molecular weight of 1.2 kDa to 10 kDa and low molecular weight thiolated hyaluronic acid with a molecular weight of 100 kDa to 300 kDa, the thiol content of the thiolated hyaluronic acid oligosaccharides and the low molecular weight thiolated hyaluronic acid are both 37.5 μmol / g to 375 μmol / g, and the total content of hyaluronic acid in the composite thiolated hyaluronic acid solution is 0.01 mg / mL to 30 mg / mL.

[0013] In the present invention, the main function of the composite thiolated hyaluronic acid is to remove harmful free radicals in the skin at multiple levels and repair the skin barrier; the extract of alpine edelweiss (also known as edelweiss) contains edelweiss acid, chlorogenic acid, phytosterols, amino acids, etc., among which edelweiss acid is the most important antioxidant component, which can promote collagen synthesis, activate the mitochondrial network, and promote skin self-repair; in addition, it can also upregulate the expression of glutamine transaminase TG1 involucrin, loricrin and filaggrin in epidermal cells, thereby strengthening the skin barrier; the alpine scutellaria extract is rich in baicalin and apigenin-7-O-glucuronide, among which baicalin plays the role of resisting lipid peroxidation, anti-inflammatory, and reducing neuroinflammation, and apigenin-7-O-glucuronide plays the role of promoting collagen synthesis. Removing harmful free radicals can slow down the loss of collagen in the body and promote the repair of the skin barrier; and the repair of the barrier can resist the external environment, reduce the damage of oxidative stress to the body, and reduce inflammation. After the three components are organically combined, the composite thiolated hyaluronic acid can further promote the antioxidant effect of the alpine edelweiss extract and the alpine scutellaria extract, so that the antioxidant effect of the composition is significantly better than the sum of the antioxidant effects of the three components tested separately. The main reason may be that the composite thiolated hyaluronic acid can promote the repair and regeneration of antioxidant active substances (such as edelweiss acid and scutellaria glycosides) in the alpine edelweiss extract and the alpine scutellaria extract, and continue to play an antioxidant effect, so that its antioxidant effect is enhanced. At the same time, the antioxidant active substances (such as edelweiss acid and scutellaria glycosides) in the alpine edelweiss extract and the alpine scutellaria extract can also promote the repair and regeneration of the active thiol of the composite thiolated hyaluronic acid, and the three components repair each other to form a redox circulation system, so as to achieve a synergistic effect, thereby achieving a comprehensive antioxidant effect and an obvious antioxidant effect. The antioxidant composition of the present invention, the three components are organically combined to play an antioxidant and barrier repair effect, and is particularly suitable for sensitive skin people. It should be noted that the antioxidant composition of the present invention preferably uses water as a solvent, more preferably uses purified water as a solvent, and the amount of the solvent added is 50 to 200 parts by mass.

[0014] In the present invention, the thiolated hyaluronic acid oligosaccharide (polymerization degree 3-25) with a molecular weight of 1.2kDa to 10kDa can penetrate into the deep dermis more than 100 microns from the skin surface. At the same time, considering the damage to the skin epidermis caused by the harsh environment (such as small particle pollutants, ultraviolet radiation, etc.), the low molecular weight thiolated hyaluronic acid with a molecular weight of 100kDa to 300kDa can penetrate into the epidermis about 0-50 microns from the skin surface. Therefore, the composite thiolated hyaluronic acid can achieve multi-level removal of harmful free radicals in the skin; it should be noted that when the molecular weight of the thiolated hyaluronic acid oligosaccharide is too low, such as less than 1.2kDa (polymerization degree 1-2), it cannot be recognized by the receptor cells and cannot play a good role. Its biological function, when the molecular weight of thiolated hyaluronic acid is too high, for example, more than 300kDa, its absorption effect becomes poor, therefore, the composite thiolated hyaluronic acid is preferably composed of thiolated hyaluronic acid oligosaccharides with a molecular weight of 1.2kDa to 10kDa and low molecular weight thiolated hyaluronic acid with a molecular weight of 100kDa to 300kDa; in addition, although the increase of the thiolation content in thiolated hyaluronic acid is beneficial to the elimination of harmful free radicals, too high a thiol content means that more chemical modification is required, and excessive modification may affect the recognition of hyaluronic acid by cell receptors, therefore, the thiol content in the thiolated hyaluronic acid oligosaccharide is preferably 37.5μmol / g polymer to 375μmol / g polymer.

[0015] Specifically, the composite thiolated hyaluronic acid solution is 0.02-15 parts by mass or 2-10 parts by mass, 2-10 parts by mass being preferred; the alpine edelweiss extract is 0.02-15 parts by mass or 0.2-5 parts by mass, 0.2-5 parts by mass being preferred; the alpine scutellaria baicalensis extract is 0.01-7.5 parts by mass or 0.1-2.5 parts by mass, 0.1-2.5 parts by mass being preferred.

[0016] Specifically, the mass ratio of the thiolated hyaluronic acid oligosaccharide to the low molecular weight thiolated hyaluronic acid is 1:4 to 4:1.

[0017] Specifically, the mass ratio of the composite thiolated hyaluronic acid solution to the sum of the mass of the alpine edelweiss extract and the alpine scutellaria baicalensis extract is 1:15 to 10:1.

[0018] Specifically, the mass ratio of the alpine edelweiss extract to the alpine scutellaria extract is 5:1 to 1:5 or 2:1 to 1:2, and 2:1 to 1:2 is a preferred mass ratio.

[0019] Specifically, the total content of hyaluronic acid in the composite thiolated hyaluronic acid solution is 2 mg / mL to 10 mg / mL.

[0020] To solve the above technical problems, the second aspect of the present invention is to provide an application of the aforementioned antioxidant composition in the preparation of antioxidant cosmetics. Since the composite thiolated hyaluronic acid in the aforementioned antioxidant composition contains two molecular segments of thiolated hyaluronic acid, it can penetrate into different depths of the skin, and the active thiol groups it carries can achieve full-layer anti-oxidation by removing harmful free radicals; the alpine edelweiss extract in the aforementioned antioxidant composition has an antioxidant effect, which can promote collagen synthesis, activate the mitochondrial network, and promote skin self-repair; the scutellaria extract in the aforementioned antioxidant composition has the effects of anti-lipid peroxidation, anti-inflammatory, and reducing neuroinflammation. The three ingredients are organically combined to exert antioxidant and barrier repair effects, which are especially suitable for sensitive skin people. Therefore, it is suitable for use in the preparation of antioxidant cosmetics. For example, the antioxidant composition of the present invention can be combined with moisturizers, allergy-relieving agents, skin conditioners, antioxidants, thickeners, emulsifiers, preservatives, acid-base regulators, chelating agents and other auxiliary materials and / or other active ingredients to obtain antioxidant cosmetics. In the present invention, the antioxidant cosmetics may be, for example, facial masks, essence water, essence, lotion, facial cream, eye cream, lip essence, body lotion, and shower gel.

[0021] In order to solve the above technical problems, the third aspect of the present invention is to provide a method for preparing the above antioxidant composition.

[0022] The steps include:

[0023] Step S1, dissolving a hyaluronic acid oligosaccharide raw material with a molecular weight of 10 kDa to 180 kDa in water, adding hydrochloric acid for acid hydrolysis, and obtaining a first acid hydrolysis solution;

[0024] Step S2, dissolving a low molecular weight hyaluronic acid raw material with a molecular weight of 300 kDa to 500 kDa in water, adding hydrochloric acid for acid hydrolysis, and obtaining a second acid hydrolysis solution;

[0025] Step S3, mixing the first acid hydrolysis solution and the second acid hydrolysis solution, and performing thiol modification on the mixed solution, wherein the thiol content after modification is 37.5 μmol / g to 375 μmol / g;

[0026] Step S4, placing the thiolated mixed system obtained in step S3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, collecting the feed liquid in the dialysis bag, and adding purified water to adjust the total hyaluronic acid content to 0.01 mg / mL to 30 mg / mL, to obtain a composite thiolated hyaluronic acid solution;

[0027] Step S5, taking the complex thiolated hyaluronic acid solution obtained in step S4, mixing it with the alpine edelweiss extract and the alpine scutellaria extract to obtain an antioxidant composition.

[0028] Specifically, in step S1, the hyaluronic acid oligosaccharide raw material is sodium hyaluronate, and the concentration of hydrochloric acid is 4 to 8 mol / L;

[0029] In step S2, the low molecular weight hyaluronic acid raw material is sodium hyaluronate, and the concentration of hydrochloric acid is 4-8 mol / L.

[0030] Specifically, in step S3, the first acid hydrolysis solution and the second acid hydrolysis solution are mixed in a mass ratio of 1:4 to 4:1.

[0031] Specifically, in step S4, the total content of hyaluronic acid in the composite thiolated hyaluronic acid solution is 2 mg / mL to 10 mg / mL.

[0032] Specifically, in step S5, the composite thiolated hyaluronic acid solution is 0.02 to 15 parts by mass or 2 to 10 parts by mass, and 2 to 10 parts by mass is preferred; the alpine edelweiss extract is 0.02 to 15 parts by mass or 0.2 to 5 parts by mass, and 0.2 to 5 parts by mass is preferred; the alpine scutellaria baicalensis extract is 0.01 to 7.5 parts by mass or 0.1 to 2.5 parts by mass, and 0.1 to 2.5 parts by mass is preferred; the preferred solvent is water, and the more preferred solvent is purified water, and the amount of solvent added is 50 to 200 parts by mass.

[0033] Specifically, in step S5, the mass ratio of the composite thiolated hyaluronic acid solution to the sum of the mass of the alpine edelweiss extract and the alpine scutellaria baicalensis extract is 1:15 to 10:1;

[0034] The mass ratio of the alpine edelweiss extract to the alpine scutellaria extract is 5:1 to 1:5 or 2:1 to 1:2, and 2:1 to 1:2 is the preferred mass ratio.

[0035] The preparation method of the aforementioned antioxidant composition provided by the present invention is simple to operate and easy to industrialize. It should be noted that the thiol modification of step S3 can adopt the method disclosed by Shu et al. (Shu et al., Biomacromolecules, 2002, 3: 1304-1311), specifically, the mixed acid hydrolyzate is reacted with a diamine containing a disulfide bond under the action of a catalyst carbodiimide and an activating reagent N-hydroxysulfosuccinimide, and finally a reducing agent is added to reduce the disulfide bond to a free thiol group to obtain a thiolated hyaluronic acid solution. In addition, the alpine edelweiss extract and the alpine scutellaria extract in the present invention are both commercially available. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 These are the test results of the zebrafish embryo antioxidant effect test. DETAILED DESCRIPTION

[0038] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] Step 1: 1.0 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the first acid hydrolysis solution, which was set aside;

[0041] Step 2: 1.0 g of sodium hyaluronate (MW 300 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the second acid hydrolysis solution for standby use;

[0042] Step 3: Mix the first acid hydrolyzate and the second acid hydrolyzate evenly, add 0.24g Sulfo-NHS and 1.8g dithiodipropionic acid dihydrazide (DTP), then add 1.08g EDCI solid, stir to dissolve, add an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at the aforementioned specific value, and stir the reaction overnight. Add 1.0N sodium hydroxide, adjust the pH value of the reaction solution to 8.5, and terminate the reaction. Then add 1.0g dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0N hydrochloric acid, adjust the pH value of the above reaction solution to 3.0, and obtain a thiolated hyaluronic acid solution. The total hyaluronic acid content is 8.33mg / ml after sampling and detection;

[0043] Step 4: The thiolated hyaluronic acid solution obtained in step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, the liquid in the dialysis bag is collected, purified water is added to adjust the total content of hyaluronic acid to 7.0 mg / ml, and the thiol content of the sample is 50.3 μmol / g polymer;

[0044] Step 5: Take 5 g of the composite thiolated hyaluronic acid solution obtained in step 4, mix it with 2.5 g of alpine edelweiss extract and 1.25 g of alpine scutellaria extract, then add purified water to 100 g, and stir evenly;

[0045] Step 6: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 5, stir evenly, and obtain an antioxidant cosmetic. Similarly, repeat steps 5 and 6 to prepare any amount of antioxidant cosmetics.

[0046] Example 2

[0047] Step 1: 1.0 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the first acid hydrolysis solution, which was set aside;

[0048] Step 2: 1.0 g of sodium hyaluronate (MW 300 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the second acid hydrolysis solution for standby use;

[0049] Step 3: Mix the first acid hydrolyzate and the second acid hydrolyzate evenly, add 0.72g of Sulfo-NHS and 3.6g of dithiodipropionic acid dihydrazide (DTP), then add 2.16g of EDCI solid, stir to dissolve, add an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at the aforementioned specific value, and stir the reaction overnight. Add 1.0N sodium hydroxide, adjust the pH value of the reaction solution to 8.5, and terminate the reaction. Then add 1.0g of dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0N hydrochloric acid, adjust the pH value of the above reaction solution to 3.0, and obtain a thiolated hyaluronic acid solution. The total content of hyaluronic acid detected by sampling is 8.09mg / ml;

[0050] Step 4: The thiolated hyaluronic acid solution obtained in step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, the liquid in the dialysis bag is collected, purified water is added to adjust the total content of hyaluronic acid to 7.0 mg / ml, and the thiol content is sampled and detected to be 252.9 μmol / g polymer;

[0051] Step 5: Take 5 g of the composite thiolated hyaluronic acid solution obtained in step 4, mix it with 2.5 g of alpine edelweiss extract and 1.25 g of alpine scutellaria extract, add purified water to 100 g, and stir evenly;

[0052] Step 6: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 5, stir evenly, and obtain an antioxidant cosmetic. Similarly, repeat steps 5 and 6 to prepare any amount of antioxidant cosmetics.

[0053] Example 3

[0054] Step 1: 0.4 g of sodium hyaluronate (MW 10 kDa) was dissolved in 40 ml of distilled water, 1.2 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the first acid hydrolysis solution, which was set aside;

[0055] Step 2: 1.6 g of sodium hyaluronate (MW 300 kDa) was dissolved in 160 ml of distilled water, 4.8 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the second acid hydrolysis solution, which was set aside;

[0056] Step 3: Mix the first acid hydrolyzate and the second acid hydrolyzate evenly, add 0.24g Sulfo-NHS and 1.8g dithiodipropionic acid dihydrazide (DTP), then add 1.08g EDCI solid, stir to dissolve, add an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at the aforementioned specific value, and stir the reaction overnight. Add 1.0N sodium hydroxide, adjust the pH value of the reaction solution to 8.5, and terminate the reaction. Then add 1.0g dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0N hydrochloric acid, adjust the pH value of the above reaction solution to 3.0, and obtain a thiolated hyaluronic acid solution. The total hyaluronic acid content is 8.29mg / ml after sampling and detection;

[0057] Step 4: The thiolated hyaluronic acid solution obtained in step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, the liquid in the dialysis bag is collected, purified water is added to adjust the total content of hyaluronic acid to 7.0 mg / ml, and the thiol content is sampled and tested to be 51.0 μmol / g polymer;

[0058] Step 5: Take 5 g of the composite thiolated hyaluronic acid solution obtained in step 4, mix it with 2.5 g of alpine edelweiss extract and 1.25 g of alpine scutellaria extract, add purified water to 100 g, and stir evenly;

[0059] Step 6: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 5, stir evenly, and obtain an antioxidant cosmetic. Similarly, repeat steps 5 and 6 to prepare any amount of antioxidant cosmetics.

[0060] Example 4

[0061] Step 1: 1.6 g of sodium hyaluronate (MW 10 kDa) was dissolved in 160 ml of distilled water, 4.8 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the first acid hydrolysis solution, which was set aside;

[0062] Step 2: 0.4 g of sodium hyaluronate (MW 300 kDa) was dissolved in 40 ml of distilled water, 1.2 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the second acid hydrolysis solution, which was set aside;

[0063] Step 3: Mix the first acid hydrolyzate and the second acid hydrolyzate evenly, add 0.24g Sulfo-NHS and 1.8g dithiodipropionic acid dihydrazide (DTP), then add 1.08g EDCI solid, stir to dissolve, add an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at the aforementioned specific value, and stir the reaction overnight. Add 1.0N sodium hydroxide, adjust the pH value of the reaction solution to 8.5, and terminate the reaction. Then add 1.0g dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0N hydrochloric acid, adjust the pH value of the above reaction solution to 3.0, and obtain a thiolated hyaluronic acid solution. The total content of hyaluronic acid detected by sampling is 8.21mg / ml;

[0064] Step 4: The thiolated hyaluronic acid solution obtained in step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, the liquid in the dialysis bag is collected, purified water is added to adjust the total content of hyaluronic acid to 7.0 mg / ml, and the thiol content is sampled and tested to be 50.0 μmol / g polymer;

[0065] Step 5: Take 5 g of the composite thiolated hyaluronic acid solution obtained in step 4, mix it with 2.5 g of alpine edelweiss extract and 1.25 g of alpine scutellaria extract, add purified water to 100 g, and stir evenly;

[0066] Step 6: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 5, stir evenly, and obtain an antioxidant cosmetic. Similarly, repeat steps 5 and 6 to prepare any amount of antioxidant cosmetics.

[0067] Embodiments 5 to 14

[0068] Examples 5 to 14: The preparation method is the same as that of Example 1, except that the addition amounts of the composite thiolated hyaluronic acid solution, alpine edelweiss extract, and alpine scutellaria extract in the antioxidant composition are different. The thiol content (50.3 μmol / g polymer) remains unchanged, the mass ratio of the thiolated hyaluronic acid of the two-band molecular weight (1:1) remains unchanged, and the addition amounts of p-hydroxyacetophenone (0.5 g) and 1,2-hexanediol (0.5 g) remain unchanged. The addition amounts of the composite thiolated hyaluronic acid solution, alpine edelweiss extract, and alpine scutellaria extract of Examples 5 to 14 are detailed in Table 1; similarly, in Examples 5 to 14, antioxidant cosmetics of any mass can be prepared by scaling up or down in the same proportion.

[0069] Table 1

[0070]

[0071] Comparative Example 1

[0072] Step 1: 1.0 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the first acid hydrolysis solution, which was set aside;

[0073] Step 2: 1.0 g of sodium hyaluronate (MW 300 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 7.0, to obtain the second acid hydrolysis solution for standby use;

[0074] Step 3: The first acid hydrolyzate and the second acid hydrolyzate are mixed evenly, and placed into a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, and the feed liquid in the dialysis bag is collected;

[0075] Step 4: Add purified water to adjust the total hyaluronic acid content in the composite thiolated hyaluronic acid solution obtained in step 3 to 7.0 mg / ml, take 5 g of the composite thiolated hyaluronic acid solution, mix it with 2.5 g of alpine edelweiss extract and 1.25 g of alpine scutellaria extract, add purified water to 100 g, and stir evenly;

[0076] Step 5: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 4 and stir evenly to obtain a cosmetic reference substance. By scaling up or down in the same proportion, any mass of cosmetic reference substance can be prepared.

[0077] Comparative Example 2

[0078] Step 1: 2.0 g of sodium hyaluronate (MW 10 kDa) was dissolved in 200 ml of distilled water, 6 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain an acid hydrolysis solution for later use;

[0079] Step 2: Add 0.24g Sulfo-NHS and 1.8g dithiodipropionic acid dihydrazide (DTP) to the acid hydrolysis solution, then add 1.08g EDCI solid, stir to dissolve, add an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at the aforementioned specific value, and stir the reaction overnight. Add 1.0N sodium hydroxide, adjust the pH value of the reaction solution to 8.5, and terminate the reaction. Then add 1.0g dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0N hydrochloric acid, adjust the pH value of the above reaction solution to 3.0, and obtain a thiolated hyaluronic acid solution. The total hyaluronic acid content is 8.20mg / ml after sampling;

[0080] Step 3: The thiolated hyaluronic acid solution obtained in step 2 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, the liquid in the dialysis bag is collected, purified water is added to adjust the total hyaluronic acid content to 7.0 mg / ml, and the thiol content is 52.2 μmol / g polymer after sampling and detection;

[0081] Step 4: Take 5 g of the product obtained in step 3, mix it with 2.5 g of alpine edelweiss extract and 1.25 g of alpine scutellaria extract, add purified water to 100 g, and stir evenly;

[0082] Step 5: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 4 and stir evenly to obtain a cosmetic reference substance. By scaling up or down in the same proportion, any mass of cosmetic reference substance can be prepared.

[0083] Comparative Example 3

[0084] Step 1: 2.0 g of sodium hyaluronate (MW 300 kDa) was dissolved in 200 ml of distilled water, 6 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain an acid hydrolysis solution for later use;

[0085] Step 2: Add 0.24g of Sulfo-NHS and 1.8g of dithiodipropionic acid dihydrazide (DTP) to the acid hydrolysis solution, then add 1.08g of EDCI solid, stir to dissolve, add an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at the aforementioned specific value, and stir to react overnight. Add 1.0N sodium hydroxide, adjust the pH value of the reaction solution to 8.5, and terminate the reaction. Then add 1.0g of dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0N hydrochloric acid, adjust the pH value of the above reaction solution to 3.0, and obtain a thiolated hyaluronic acid solution. The total content of hyaluronic acid detected by sampling is 8.44mg / ml;

[0086] Step 3: The thiolated hyaluronic acid solution obtained in step 2 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, the liquid in the dialysis bag is collected, purified water is added to adjust the total content of hyaluronic acid to 7.0 mg / ml, and the thiol content is sampled and detected to be 49.8 μmol / g polymer;

[0087] Step 4: Take 5 g of the product obtained in step 3, mix it with 2.5 g of alpine edelweiss extract and 1.25 g of alpine scutellaria extract, add purified water to 100 g, and stir evenly;

[0088] Step 5: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 4 and stir evenly to obtain a cosmetic reference substance. By scaling up or down in the same proportion, any mass of cosmetic reference substance can be prepared.

[0089] Comparative Example 4

[0090] Step 1: 1.0 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the first acid hydrolysis solution, which was set aside;

[0091] Step 2: 1.0 g of sodium hyaluronate (MW 300 kDa) was dissolved in 100 ml of distilled water, 3 ml of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40°C for 10 minutes. An appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to adjust the solution to a specific pH value, the pH value of which was 4.75, to obtain the second acid hydrolysis solution for standby use;

[0092] Step 3: Mix the first acid hydrolyzate and the second acid hydrolyzate evenly, add 0.24g Sulfo-NHS and 1.8g dithiodipropionic acid dihydrazide (DTP), then add 1.08g EDCI solid, stir to dissolve, add an appropriate amount of acid or base (0.1N hydrochloric acid or sodium hydroxide) to keep the pH value of the reaction solution stable at the aforementioned specific value, and stir the reaction overnight. Add 1.0N sodium hydroxide, adjust the pH value of the reaction solution to 8.5, and terminate the reaction. Then add 1.0g dithiothreitol, stir to dissolve, and react for 4 hours. Then add 1.0N hydrochloric acid, adjust the pH value of the above reaction solution to 3.0, and obtain a thiolated hyaluronic acid solution. The total hyaluronic acid content is 8.33mg / ml after sampling and detection;

[0093] Step 4: The thiolated hyaluronic acid solution obtained in step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, the liquid in the dialysis bag is collected, purified water is added to adjust the total content of hyaluronic acid to 7.0 mg / ml, and the thiol content of the sample is 50.3 μmol / g polymer;

[0094] Step 5: Take 5 g of the product obtained in step 4 and add purified water to 100 g;

[0095] Step 6: Add 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol to the product prepared in step 5, stir evenly, and obtain an antioxidant cosmetic. Similarly, repeat steps 5 and 6 to prepare any amount of antioxidant cosmetics.

[0096] Comparative Example 5

[0097] Step 1: Take 2.5g of alpine edelweiss extract and add purified water to 100g, mix well, then add 0.5g of p-hydroxyacetophenone and 0.5g of 1,2-hexanediol, stir well to obtain a cosmetic reference substance; by enlarging or reducing the same proportion, any mass of cosmetic reference substance can be prepared.

[0098] Comparative Example 6

[0099] Step 1: Take 1.25g of Scutellaria baicalensis extract and add purified water to 100g, mix well, then add 0.5g of p-hydroxyacetophenone and 0.5g of 1,2-hexanediol, stir well to obtain the cosmetic reference substance; by enlarging or reducing the same proportion, any mass of cosmetic reference substance can be prepared.

[0100] Comparative Example 7

[0101] Step 1: Take 2.5g of alpine edelweiss extract and 1.25g of alpine scutellaria extract, add purified water to 100g, mix evenly, then add 0.5g of p-hydroxyacetophenone and 0.5g of 1,2-hexanediol, stir evenly to obtain a cosmetic reference substance; by enlarging or reducing the same proportion, any mass of cosmetic reference substance can be prepared.

[0102] Test Example 1 In vitro antioxidant test

[0103] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH for short) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517nm. When there is a free radical scavenger, the light absorption of DPPH ethanol solution is weakened due to pairing with its single electron. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the size of the antioxidant activity.

[0104] The DPPH free radical scavenging rate was calculated as follows:

[0105]

[0106] Where:

[0107] T—absorbance value of sample tube, i.e. absorbance value of solution after reaction between sample and DPPH;

[0108] T 0 —Sample background absorbance;

[0109] C—the average value of the absorbance of the DPPH tube three times, that is, the absorbance of the DPPH solution when no sample is added;

[0110] C 0 —Solvent background absorbance.

[0111] The antioxidant cosmetics prepared in Examples 1 to 14, the cosmetic reference substances prepared in Comparative Examples 1 to 6, and 1 kDa and 200 kDa common hyaluronic acid samples were selected, and distilled water was added to prepare a total content of complex thiolated hyaluronic acid or common hyaluronic acid content of 0.2 mg / mL, and a DPPH free radical scavenging experiment was performed. The results are shown in Table 2.

[0112] Table 2

[0113]

[0114]

[0115] It can be seen from Table 2 that the antioxidant cosmetics prepared by Examples 1-14 show that the hyaluronic acid has a significant ability to scavenge free radicals after being thiol-modified; it also shows that the antioxidant cosmetics prepared by Examples 1 to 14 can effectively scavenge free radicals; it further shows that the oxidative efficacy of the composite thiol-modified hyaluronic acid solution, the alpine edelweiss extract and the alpine scutellaria extract is synergistic, and the antioxidant efficacy of the antioxidant composition of the present invention is better than the sum of the antioxidant effects of each of the compositions tested separately.

[0116] Test Example 2 Antioxidant Effect Test on Zebrafish Embryos

[0117] 1. Test principle: The reagent is a DNA dye with weak fluorescence itself. It can be oxidized by oxygen free radicals (ROS) in the cell. The oxidation products combine with DNA to produce bright green fluorescence. It is soluble in organic solvents (such as dimethyl sulfoxide), and the main component of zebrafish yolk sac is fat. The permeability of the yolk sac is strong, and the staining is obvious. Menadione can produce active oxygen free radicals. When the amount of free radicals produced is greater than the body's clearance capacity, oxidative stress occurs. The fluorescence intensity of the zebrafish yolk sac is used to evaluate whether the sample has an antioxidant effect.

[0118] 2. Test method:

[0119] 1) Group setting: zebrafish were randomly selected in 8-well plates, with 15 zebrafish in each well. Two wells (30 fish in total) were selected as the normal control group, two wells (30 fish in total) were selected as the model control group, two wells (30 fish in total) were selected as the experimental group, and two wells (30 fish in total) were selected as the comparative group.

[0120] 2) Feeding method: The model control group, the experimental group and the comparative example group were given menadione to establish a zebrafish oxidative stress model, wherein the experimental group was given the sample prepared in Example 1 of the present invention (distilled water was diluted to a total content of 0.2 mg / mL of composite thiolated hyaluronic acid, 3 mL per well); the comparative example group was given the sample prepared in Comparative Example 1 of the present invention (distilled water was diluted to a total content of 0.2 mg / mL of hyaluronic acid, 3 mL per well); and the model control group was not given the composition of the present invention, and was raised normally; the normal control group was not given menadione, and the zebrafish oxidative stress model was not established, nor was the composition of the present invention, and was raised normally. The feeding conditions were incubated in dark at 28 ° C for 22 h, and zebrafish were stained with a specific ROS fluorescent reagent. After the staining was completed, 10 zebrafish were randomly selected in each experimental group and placed under a fluorescence microscope for photographing, and image processing software was used to analyze and collect data, and the zebrafish yolk sac fluorescence intensity (S) was analyzed, and the sample was calculated and judged whether it had an antioxidant effect according to the formula.

[0121]

[0122] The experimental results are as follows Figure 1 As shown, it can be seen from the experimental results that the sample group of the experimental group calculated according to the above calculation formula has a zebrafish embryo reactive oxygen (ROS) scavenging rate of 85% (P < 0.001), which means that the antioxidant cosmetics prepared in Example 1 have an antioxidant effect; the sample group of the comparative example group calculated according to the above calculation formula has a zebrafish embryo reactive oxygen (ROS) scavenging rate of 14% (P < 0.001), which means that the antioxidant cosmetics prepared in Example 1 have a stronger antioxidant effect than the cosmetic reference prepared in Comparative Example 1 (P < 0.001).

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An antioxidant composition, characterized in that The components include: Complex thiolated hyaluronic acid solution; Alpine edelweiss extract; Scutellaria alpinia extract; The composite thiolated hyaluronic acid solution comprises thiolated hyaluronic acid oligosaccharides with a molecular weight of 1.2 kDa to 10 kDa and low molecular weight thiolated hyaluronic acid with a molecular weight of 100 kDa to 300 kDa, the thiol content of the thiolated hyaluronic acid oligosaccharides and the low molecular weight thiolated hyaluronic acid are both 37.5 μmol / g to 375 μmol / g, and the total content of hyaluronic acid in the composite thiolated hyaluronic acid solution is 0.01 mg / mL to 30 mg / mL.

2. The antioxidant composition according to claim 1, characterized in that The composite thiolated hyaluronic acid solution is 0.02 to 15 parts by mass or 2 to 10 parts by mass, the alpine edelweiss extract is 0.02 to 15 parts by mass or 0.2 to 5 parts by mass, and the alpine scutellaria baicalensis extract is 0.01 to 7.5 parts by mass or 0.1 to 2.5 parts by mass.

3. The antioxidant composition according to claim 1, characterized in that The mass ratio of the thiolated hyaluronic acid oligosaccharide to the low molecular weight thiolated hyaluronic acid is 1:4 to 4:

1.

4. The antioxidant composition according to claim 1, characterized in that The mass ratio of the composite thiolated hyaluronic acid solution to the sum of the mass of the alpine edelweiss extract and the alpine scutellaria baicalensis extract is 1:15 to 10:

1.

5. The antioxidant composition according to claim 1, characterized in that The mass ratio of the alpine edelweiss extract to the alpine scutellaria baicalensis extract is 5:1 to 1:5 or 2:1 to 1:

2.

6. The antioxidant composition according to claim 1, characterized in that The total content of hyaluronic acid in the composite thiolated hyaluronic acid solution is 2 mg / mL to 10 mg / mL.

7. Use of the antioxidant composition according to any one of claims 1 to 6 in the preparation of antioxidant cosmetics.

8. The method for preparing the antioxidant composition according to any one of claims 1 to 6, characterized in that: The steps include: Step S1, dissolving a hyaluronic acid oligosaccharide raw material with a molecular weight of 10 kDa to 180 kDa in water, adding hydrochloric acid for acid hydrolysis, and obtaining a first acid hydrolysis solution; Step S2, dissolving a low molecular weight hyaluronic acid raw material with a molecular weight of 300 kDa to 500 kDa in water, adding hydrochloric acid for acid hydrolysis, and obtaining a second acid hydrolysis solution; Step S3, mixing the first acid hydrolysis solution and the second acid hydrolysis solution, and performing thiol modification on the mixed solution, wherein the thiol content after modification is 37.5 μmol / g to 375 μmol / g; Step S4, placing the thiolated mixed system obtained in step S3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, collecting the feed liquid in the dialysis bag, and adding purified water to adjust the total hyaluronic acid content to 0.01 mg / mL to 30 mg / mL, to obtain a composite thiolated hyaluronic acid solution; Step S5, taking the complex thiolated hyaluronic acid solution obtained in step S4, mixing it with the alpine edelweiss extract and the alpine scutellaria extract to obtain an antioxidant composition.

9. The preparation method according to claim 8, characterized in that: In step S1, the hyaluronic acid oligosaccharide raw material is sodium hyaluronate, and the concentration of hydrochloric acid is 4-8 mol / L; In step S2, the low molecular weight hyaluronic acid raw material is sodium hyaluronate, and the concentration of hydrochloric acid is 4-8 mol / L.

10. The preparation method according to claim 8, characterized in that: In step S3, the first acid hydrolysis solution and the second acid hydrolysis solution are mixed in a mass ratio of 1:4 to 4:

1.

11. The preparation method according to claim 8, characterized in that: In step S4, the total content of hyaluronic acid in the composite thiolated hyaluronic acid solution is 2 mg / mL to 10 mg / mL.

12. The preparation method according to claim 8, characterized in that: In step S5, the composite thiolated hyaluronic acid solution is 0.02 to 15 parts by mass or 2 to 10 parts by mass, the alpine edelweiss extract is 0.02 to 15 parts by mass or 0.2 to 5 parts by mass, and the alpine scutellaria baicalensis extract is 0.01 to 7.5 parts by mass or 0.1 to 2.5 parts by mass.

13. The preparation method according to claim 8, characterized in that: In step S5, the mass ratio of the composite thiolated hyaluronic acid solution to the sum of the mass of the alpine edelweiss extract and the alpine scutellaria baicalensis extract is 1:15 to 10:1; The mass ratio of the alpine edelweiss extract to the alpine scutellaria baicalensis extract is 5:1 to 1:5 or 2:1 to 1:2.

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