Preparation method of aqueous solution hyaluronic acid oligosaccharide composition, composition and application

Abstract

The invention discloses a preparation method of an aqueous solution hyaluronic acid oligosaccharide composition, the composition and application wherein the preparation method comprises the following steps: modifying acidolyzed hyaluronic acid to obtain a sulfhydrylated hyaluronic acid solution, then mixing the sulfhydrylated hyaluronic acid solution with an acidolysis solution in a volume ratio of less than 1: 5, and then dialyzing and ultra-filtering to obtain the aqueous solution hyaluronic acid oligosaccharide composition. The total content of hyaluronic acid is 0.2 mg / mL-30 mg / mL, and the content of sulfydryl is 1 [mu] mol / g polymer to 250 [mu] mol / g polymer. The preparation method is simple to operate, the aqueous solution containing the sulfhydrylated hyaluronic acid oligosaccharide and the hyaluronic acid oligosaccharide or the salt of the hyaluronic acid oligosaccharide is prepared, the molecular weight distribution of the product is uniform, the range is proper, the product is easily absorbed by the skin, then reactive oxygen free radicals can be effectively eliminated, meanwhile, the product is not prone to oxidation cross-linking inactivation, and the product is suitable for being applied to anti-aging products.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a preparation method, a composition and an application of an aqueous solution hyaluronic acid oligosaccharide composition. 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] 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 of its own weight of water. The transdermal absorption capacity of the skin is related to the molecular weight of hyaluronic acid and its derivatives. The lower the molecular weight, the better the absorption. Low molecular weight hyaluronic acid can be better absorbed by the skin when applied externally because of its small relative molecular weight, reaching the deep dermis of the skin, promoting the proliferation and differentiation of epidermal cells, and removing active oxygen free radicals. However, the ability of such quenching active oxygen free radicals is very weak, which is not enough 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). At the same time, hyaluronic acid oligosaccharides with too low a degree of polymerization (1-2) cannot be recognized by receptor cells and cannot exert their biological functions well.

[0004] 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 remove reactive oxygen free radicals. However, in the prior art, the molecular weight of chemically modified hyaluronic acid thiol derivatives is usually between 20KDa and 2500KDa. There are two main problems with the application of such hyaluronic acid thiol derivatives in the field of skin: on the one hand, the skin absorption efficiency is reduced by too high a molecular weight, and it may even not be absorbed by the skin; on the other hand, the hyaluronic acid thiol derivatives have too high a molecular weight and are prone to lose their ability to resist reactive oxygen due to the occurrence of cross-linking caused by oxidation reactions.

[0005] Therefore, although the current hyaluronic acid skin care products have improved some symptoms of skin aging to a certain extent, they cannot effectively remove active oxygen free radicals and fail to effectively eliminate the root causes of skin aging. Hyaluronic acid thiol modification also loses the ability to remove active oxygen free radicals due to its large molecular weight and oxidative cross-linking reaction. Therefore, it is meaningful to develop a hyaluronic acid oligosaccharide composition that is easily absorbed by the skin and can maintain the activity of thiol groups, so as to exert its effect of removing active oxygen free radicals and firming and anti-wrinkle effects, and delay the aging of human skin.

[0006] In addition, there are many methods for preparing hyaluronic acid oligosaccharides. For example, patent CN106518934A uses macromolecular hyaluronic acid as raw material, and after hyaluronidase hydrolysis, heat inactivation, activated carbon filtration and spray drying, an ultra-low molecular weight hyaluronic acid product with an average molecular weight of less than 1200 Daltons can be obtained. Patent CN111040048A discloses a method for preparing unsaturated hyaluronic acid odd-numbered oligosaccharides, which mainly uses microbial hyaluronidase to enzymolyze hyaluronic acid, then separates and purifies it through a low-pressure chromatography column, and finally desalts it by size exclusion chromatography to obtain a series of unsaturated hyaluronic acid odd-numbered oligosaccharides. However, the hyaluronic acid oligosaccharides prepared by this method have limited ability to scavenge reactive oxygen free radicals. At the same time, the thiol modification of hyaluronic acid / biocompatible polymer or the preparation of disulfide cross-linked biocompatible polymer cross-linked materials mostly focus on higher molecular weights, such as CN102399295A patent discloses a class of low-modified biocompatible polymer thiol modified derivatives and their uses in medicine and pharmacy, wherein the molecular weight of the biocompatible polymer is preferably between 20000 and 1500000Da. The main purpose of these thiol modification technologies is to improve the low viscosity of ordinary hyaluronic acid and improve the in vivo anti-enzymatic performance by utilizing the thiol group to easily oxidize and cross-link into gel, therefore, its scavenging active oxygen free radicals and firming anti-wrinkle effect, delaying the aging of human skin is limited. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a preparation method, composition and application of an aqueous solution hyaluronic acid oligosaccharide composition. Through a simple preparation process, an aqueous solution comprising both thiolated hyaluronic acid oligosaccharides and hyaluronic acid oligosaccharides or their salts is prepared, and the product has a uniform molecular weight distribution and an appropriate range, is easily absorbed by the skin, and can effectively eliminate active oxygen free radicals. At the same time, it is not easy to be inactivated by oxidation and cross-linking, and is suitable for use in anti-aging products.

[0008] In order to solve the above technical problems, the first aspect of the present invention is to provide a method for preparing an aqueous solution hyaluronic acid oligosaccharide composition, comprising the following steps: Step 1, dissolving a hyaluronic acid raw material in water, then adding hydrochloric acid for acid hydrolysis to obtain an acid hydrolyzate, and then performing thiol modification on the hyaluronic acid in the acid hydrolyzate to obtain a thiol-modified hyaluronic acid solution; Step 2, dissolving the hyaluronic acid raw material in water, and then adding hydrochloric acid for acid hydrolysis to obtain the same acid hydrolysis solution as step 1; Step 3, mixing the thiolated hyaluronic acid solution obtained in step 1 and the acid hydrolyzate obtained in step 2 at a volume ratio of less than 1:5 to obtain a mixed solution; Step 4, placing the mixed solution obtained in step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, and collecting the feed liquid in the dialysis bag; Step 5: Pass the liquid obtained in step 4 through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, collect the permeate, and obtain an aqueous solution hyaluronic acid oligosaccharide composition, wherein the total hyaluronic acid content is 0.2 mg / mL to 30 mg / mL, and the thiol content is 1 μmol / g polymer to 250 μmol / g polymer.

[0009] The present invention preferably uses a hyaluronic acid raw material with a molecular weight between 4kDa and 200kDa (more preferably, the molecular weight of the hyaluronic acid raw material is 10kDa to 50kDa), firstly acid hydrolyzes it in an acidic environment, and the degradation of the reducing end oligosaccharides and the β-elimination effect occur in the hyaluronic acid molecule, thereby reducing the molecular weight of the hyaluronic acid. The lower molecular weight can improve the efficiency of thiol modification on the one hand, and improve its transdermal absorption capacity on the other hand. Then, by dialysis and filtration, a product with a molecular weight range of 1.2kDa to 4kDa (degree of polymerization 3-10) is obtained. The hyaluronic acid oligosaccharides in this molecular weight range are also beneficial to accelerate the proliferation of fibroblasts and the synthesis of collagen, promote angiogenesis reaction and wound repair; in addition, within this molecular weight range, the thiol group is more likely to undergo disulfide cross-linking, resulting in the thiolated hyaluronic acid oligosaccharide losing or weakening its ability to eliminate oxygen free radicals. Based on this, the present application adds a hyaluronic acid similar to it to the thiolated hyaluronic acid oligosaccharide to reduce or reduce the cross-linking of the thiol group, so as to prevent its cross-linking inactivation and fail to achieve the effect of removing active oxygen free radicals. It should be noted that the method of thiol modification in step 1 can be, for example, the acid hydrolysis solution after acid hydrolysis, reacting with a diamine containing a disulfide bond under the action of a catalyst carbodiimide and an activating reagent N-hydroxysulfosuccinimide, and finally adding a reducing agent to reduce the disulfide bond to a free thiol group to obtain a thiol-modified hyaluronic acid solution.

[0010] Specifically, the hyaluronic acid raw material is selected from hyaluronic acid or its salt; more specifically, the hyaluronic acid raw material is sodium hyaluronate.

[0011] Specifically, in step 1, the thiol content is preferably 5 μmol / g polymer to 100 μmol / g polymer.

[0012] Specifically, in step 1 and step 2, the concentration of hydrochloric acid is 4 mol / L to 8 mol / L, more specifically 6 mol / L; The volume of hydrochloric acid is 2% to 4%, more specifically 3%, of the volume of the hyaluronic acid solution; The acid hydrolysis time is 10 to 120 minutes.

[0013] Specifically, in step 3, the volume ratio of the thiolated hyaluronic acid solution to the acid-hydrolyzed hyaluronic acid solution is more preferably 1:6 to 1:49.

[0014] In order to solve the above technical problems, the second aspect of the present invention is to provide an aqueous solution hyaluronic acid oligosaccharide composition obtained by the above preparation method.

[0015] The aqueous solution hyaluronic acid oligosaccharide composition provided by the present invention has a molecular weight distribution range of 1.2 kDa to 4 kDa, has a strong transdermal absorption capacity, is conducive to accelerating the proliferation of fibroblasts and the synthesis of collagen, and promotes angiogenesis reactions and wound repair; its thiol content is 1 μmol / g polymer to 250 μmol / g polymer, and can effectively remove active oxygen free radicals. At the same time, due to the presence of non-modified hyaluronic acid oligosaccharides, it has a good isolation effect on the thiol hyaluronic acid molecules, reduces the probability of collision, and then effectively inhibits the cross-linking between thiol groups, so that it has a good elimination of active oxygen free radicals.

[0016] In order to solve the above technical problems, the third aspect of the present invention is to provide the application of the aforementioned aqueous solution hyaluronic acid oligosaccharide composition in the preparation of anti-aging products. For example, in the preparation of facial masks, essence water, essence milk, essence, lotion, face cream, eye cream, lip essence and body lotion, the addition amount of the aqueous solution hyaluronic acid oligosaccharide composition is preferably 0.2% to 100%, more preferably 1% to 20%; for example, moisturizers, soothing agents, skin conditioners, antioxidants, thickeners, emulsifiers, preservatives, acid-base regulators, chelating agents and other auxiliary materials and / or other active ingredients can also be added to anti-aging products. Understandably, when the anti-aging product prepared by the aqueous solution hyaluronic acid oligosaccharide composition of the present application has at least a good elimination of active oxygen free radicals, thereby playing an anti-aging role. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 is a graph showing the experimental results of the fifth test example of the present invention; Figure 2 4 is a diagram showing the experimental results of the sixth test example of the present invention. DETAILED DESCRIPTION

[0019] 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. First embodiment

[0020] Step 1: 1.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 °C for 20 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 mL of distilled water, 30 mL of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40 °C for 20 minutes, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid hydrolyzate; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: Place the mixed solution from step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then perform dialysis purification with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is no longer visible. Finally, complete the dialysis with 0.001 N hydrochloric acid for dialysis desalination, and collect the feed solution in the dialysis bag; Step 5: The feed solution of step 4 is passed through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and the permeate is collected, which is an aqueous solution hyaluronic acid oligosaccharide composition. The total hyaluronic acid content is 10.15 mg / mL and the thiol content is 36.9 μmol / g polymer. Second embodiment

[0021] Step 1: 1.5 g of sodium hyaluronate (MW50kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 °C for 60 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 50 kDa) was dissolved in 100 mL of distilled water, 30 mL of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40 °C for 60 minutes, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid hydrolyzate; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: Place the mixed solution from step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then perform dialysis purification with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is no longer visible. Finally, complete the dialysis with 0.001 N hydrochloric acid for dialysis desalination, and collect the feed solution in the dialysis bag; Step 5: The feed solution of step 4 is passed through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and the permeate is collected, which is an aqueous solution hyaluronic acid oligosaccharide composition. The total hyaluronic acid content is 9.98 mg / mL and the thiol content is 35.8 μmol / g polymer. Third embodiment

[0022] Step 1: 1.5 g of sodium hyaluronate (MW200kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 °C for 120 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 200 kDa) was dissolved in 100 mL of distilled water, 30 mL of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40 °C for 120 minutes, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid hydrolyzate; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: Place the mixed solution from step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then perform dialysis purification with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is no longer visible. Finally, complete the dialysis with 0.001 N hydrochloric acid for dialysis desalination, and collect the feed solution in the dialysis bag; Step 5: The feed solution of step 4 is passed through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and the permeate is collected, which is an aqueous solution hyaluronic acid oligosaccharide composition. The total hyaluronic acid content is 9.73 mg / mL and the thiol content is 34.4 μmol / g polymer.

[0023] Fourth to Sixth Embodiments The preparation methods of the fourth to sixth embodiments are the same as those of the first embodiment, except that the ratios of the thiolated hyaluronic acid solution and the acid hydrolyzate in step 3 are different. The ratios and test results of the fourth to sixth embodiments are shown in Table 1.

[0024] Table 1 Seventh embodiment

[0025] Step 1: 1.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 ° C for 20 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.72 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 mL of distilled water, 30 mL of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40 °C for 20 minutes, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid hydrolyzate; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: Place the mixed solution from step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then perform dialysis purification with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is no longer visible. Finally, complete the dialysis with 0.001 N hydrochloric acid for dialysis desalination, and collect the feed solution in the dialysis bag; Step 5: The feed solution of step 4 is passed through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and the permeate is collected, which is an aqueous solution hyaluronic acid oligosaccharide composition. The total hyaluronic acid content is 10.10 mg / mL and the thiol content is 75.3 μmol / g polymer. Eighth embodiment

[0026] Step 1: 0.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 ° C for 20 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 0.5 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 mL of distilled water, 30 mL of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40 °C for 20 minutes, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid hydrolyzate; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: Place the mixed solution from step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then perform dialysis purification with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is no longer visible. Finally, complete the dialysis with 0.001 N hydrochloric acid for dialysis desalination, and collect the feed solution in the dialysis bag; Step 5: The feed solution of step 4 is passed through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and the permeate is collected, which is an aqueous solution hyaluronic acid oligosaccharide composition. The total hyaluronic acid content is 2.32 mg / mL and the thiol content is 38.4 μmol / g polymer.

[0027] The first comparison Step 1: 1.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 °C for 20 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 of 4.75. 0.09 g of Sulfo-NHS and 0.84 g of cystamine dihydrochloride were added, and then 0.18 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: The thiolated hyaluronic acid solution of step 1 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then dialyzed and purified with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride, and the purification process of the final product is tracked by gel permeation chromatography (GPC) until the impurity absorption peak is no longer visible. Finally, dialysis is completed by desalting with 0.001 N hydrochloric acid, and the liquid in the dialysis bag is collected; Step 3: The feed solution of step 2 is passed through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and the permeate is collected, which is the hyaluronic acid oligosaccharide composition of the first comparative example. The total hyaluronic acid content is 10.06 mg / mL and the thiol content is 36.2 μmol / g polymer.

[0028] The second comparison Step 1: 1.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 °C for 20 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: The thiolated hyaluronic acid solution obtained in step 1 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then dialyzed and purified with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. The purification process of the final product is tracked by gel permeation chromatography (GPC) until the impurity absorption peak is no longer visible. Finally, 0.001 N hydrochloric acid is used for dialysis desalination to complete the dialysis, and the feed liquid in the dialysis bag is collected; Step 3: Pass the feed liquid through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and collect the permeate, which is the hyaluronic acid oligosaccharide composition of the second comparative example. Sampling and detection of the total hyaluronic acid content in the hyaluronic acid oligosaccharide composition is 10.12 mg / mL, and the thiol content is 470.2 μmol / g polymer.

[0029] The third comparison Step 1: 1.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 °C for 20 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 mL of distilled water, 30 mL of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40 °C for 20 minutes, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid hydrolyzate; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:2; Step 4: Place the mixed solution from step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then perform dialysis purification with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is no longer visible. Finally, complete the dialysis with 0.001 N hydrochloric acid for dialysis desalination, and collect the feed solution in the dialysis bag; Step 5: The feed solution of step 4 is passed through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and the permeate is collected, which is the aqueous solution hyaluronic acid oligosaccharide composition of the third comparative example. The total hyaluronic acid content is 10.09 mg / mL and the thiol content is 151.7 μmol / g polymer.

[0030] The fourth comparison Step 1: 1.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, 3 mL of 6 mol / L hydrochloric acid was added, and the solution was acidified at 40 °C for 20 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 mL of distilled water, 30 mL of 6 mol / L hydrochloric acid was added, and acid hydrolysis was performed at 40 °C for 20 minutes, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid hydrolyzate; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:4; Step 4: Place the mixed solution from step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then perform dialysis purification with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride. Use gel permeation chromatography (GPC) to track the purification process of the final product until the impurity absorption peak is no longer visible. Finally, complete the dialysis with 0.001 N hydrochloric acid for dialysis desalination, and collect the feed solution in the dialysis bag; Step 5: Pass the feed solution of step 4 through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, and collect the permeate, which is the aqueous solution hyaluronic acid oligosaccharide composition of the fourth comparative example. The total hyaluronic acid content is 10.15 mg / mL and the thiol content is 74.9 μmol / g polymer.

[0031] Fifth comparison Step 1: 1.5 g of sodium hyaluronate (MW 10kDa) was dissolved in 100 mL of distilled water, and 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 10 kDa) was dissolved in 100 mL of distilled water, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid solution; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: The mixed solution of step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then dialyzed and purified with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride, and the purification process of the final product is tracked by gel permeation chromatography (GPC) until the impurity absorption peak is no longer visible. Finally, dialysis is completed by dialysis and desalination with 0.001 N hydrochloric acid, and the feed liquid in the dialysis bag is collected, which is the fifth comparative example aqueous solution hyaluronic acid oligosaccharide composition, and the total hyaluronic acid content is 13.32 mg / mL and the thiol content is 37.4 μmol / g polymer.

[0032] Sixth comparison Step 1: 1.5 g of sodium hyaluronate (MW 50 kDa) was dissolved in 100 mL of distilled water, and 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 50 kDa) was dissolved in 100 mL of distilled water, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid solution; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: The mixed solution of step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then dialyzed and purified with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride, and the purification process of the final product is tracked by gel permeation chromatography (GPC) until the impurity absorption peak is no longer visible. Finally, 0.001 N hydrochloric acid is used for dialysis and desalination to complete the dialysis, and the feed liquid in the dialysis bag is collected, which is the sixth comparative example aqueous solution hyaluronic acid oligosaccharide composition, and the total hyaluronic acid content is 13.77 mg / mL and the thiol content is 37.9 μmol / g polymer.

[0033] Seventh comparison Step 1: 1.5 g of sodium hyaluronate (MW 200kDa) was dissolved in 100 mL of distilled water, and 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 of 4.75. 0.18 g of Sulfo-NHS and 1.68 g of cystamine dihydrochloride were added, and then 0.36 g of EDCI solid was added, stirred and dissolved, and an appropriate amount of acid or base (0.1 N hydrochloric acid or sodium hydroxide) was added to keep the pH value of the reaction solution stable at the aforementioned specific value, and the reaction was stirred overnight. 1.0 N sodium hydroxide was added to adjust the pH of the reaction solution to 8.5 to terminate the reaction. Then 0.75 g of dithiothreitol was added, stirred and dissolved, and the reaction was allowed to react for 4 hours. Then 1.0 N hydrochloric acid was added to adjust the pH value of the above reaction solution to 3.0 to obtain a thiolated hyaluronic acid solution; Step 2: 1.5 g of sodium hyaluronate (MW 200 kDa) was dissolved in 100 mL of distilled water, and then 1.0 N hydrochloric acid was added to adjust the solution to a specific pH of 3.0 to obtain an acid solution; Step 3: Mix the thiolated hyaluronic acid solution of step 1 and the acid hydrolyzate of step 2 in a mass ratio of 1:9; Step 4: The mixed solution of step 3 is placed in a dialysis bag with a molecular weight cutoff of 1.2 kDa, and then dialyzed and purified with a mixed solution of 0.001 N hydrochloric acid and 0.2 N sodium chloride, and the purification process of the final product is tracked by gel permeation chromatography (GPC) until the impurity absorption peak is no longer visible. Finally, 0.001 N hydrochloric acid is used for dialysis and desalination to complete the dialysis, and the feed liquid in the dialysis bag is collected, which is the seventh comparative example aqueous solution hyaluronic acid oligosaccharide composition, and the total hyaluronic acid content is 14.25 mg / mL and the thiol content is 33.2 μmol / g polymer.

[0034] The first test case is in vitro antioxidant test 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.

[0035] The DPPH free radical scavenging rate was calculated as follows:

[0036] Where: T—absorbance of sample tube, i.e. absorbance of solution after sample reacts with DPPH; T0—sample background absorbance; 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; C0—Solvent background absorbance.

[0037] The aqueous solution hyaluronic acid oligosaccharide compositions prepared in the first to eighth embodiments and 0.8 kDa and 10 kDa ordinary hyaluronic acid samples were selected and distilled water was added to prepare active materials (Extra-MSC) with total hyaluronic acid contents of 0.25 mg / mL, 1.0 mg / mL and 2.0 mg / mL, respectively, and DPPH free radical scavenging experiments were carried out. The results are shown in Table 2.

[0038] Table 2

[0039] As can be seen from Table 2, the DPPH free radical scavenging rate of the Extra-MSC prepared by the aqueous solution hyaluronic acid oligosaccharide composition prepared by the first to eighth embodiments is about 40 to 161 times higher than that of 0.8k Da hyaluronic acid, and about 44 to 179 times higher than that of 10k Da hyaluronic acid, indicating that the thiol groups contained in the Extra-MSC prepared by the aqueous solution hyaluronic acid oligosaccharide composition prepared by the first to eighth embodiments can effectively scavenge free radicals and have good antioxidant effects.

[0040] 2',7'-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) has no spontaneous fluorescence and can freely pass through the cell membrane. After entering the cell, it is hydrolyzed by esterase to form DCFH, which cannot penetrate the cell membrane and accumulates in the cell. The reactive oxygen species in the cell can oxidize the non-fluorescent DCFH to generate fluorescent DCF. The green fluorescence intensity is proportional to the level of reactive oxygen in the cell. Hydrogen peroxide (H2O2), ultraviolet rays, blue light, etc., as exogenous oxidants, can induce an increase in the ROS content in human keratinocytes. The ROS inhibition rate is calculated by comparing the changes in the fluorescence intensity of the intracellular fluorescent dye DCFH-DA after administration of the negative control and the test substance.

[0041] The calculation formula of ROS inhibition rate is:

[0042] T—the average of three fluorescence intensities of the tested sample; C—average of three times of fluorescence intensity of negative control; C0—the average of three fluorescence intensities of the naked cell group.

[0043] The aqueous solution hyaluronic acid oligosaccharide compositions prepared in the first to eighth embodiments and 0.8 kDa and 10 kDa common hyaluronic acid samples were selected and distilled water was added to prepare Extra-MSC with a total hyaluronic acid content of 1.0 mg / mL. In vitro keratinocyte reactive oxygen species (ROS) inhibition test was performed. The test results are shown in Table 3.

[0044] Table 3

[0045] As can be seen from Table 3, the Extra-MSC prepared by the aqueous solution hyaluronic acid oligosaccharide composition prepared in the first to eighth embodiments has an inhibition rate of human epidermal keratinocytes greater than 80% (SD), and has the effect of inhibiting ROS, and the inhibition effect is better than that of the 0.8 kDa HA group and the 10 kDa HA group.

[0046] The third test example is the thiol retention test The aqueous solution hyaluronic acid oligosaccharide compositions prepared in the first to eighth embodiments and the samples prepared in the first to seventh comparative examples were selected and placed at room temperature. The thiol content was tested on the 0th, 15th, 30th and 60th days, respectively. The test results are shown in Table 4.

[0047] Table 4

[0048] It can be seen from Table 4 that the active thiol groups in the aqueous solution hyaluronic acid oligosaccharide compositions prepared by the first to eighth embodiments are stable for at least 60 days, indicating that they have good anti-aging effects and are easy to store; while the active thiol groups in the samples prepared by the first to seventh comparative examples are less stable, and their contents gradually decrease significantly with the test time, and as the storage time increases, the aqueous solution gradually gels, indicating that their anti-aging effects are limited and they are not easy to store.

[0049] Test Case 4: In vitro diffusion cell transdermal absorption test The aqueous solution hyaluronic acid oligosaccharide compositions prepared in the first to eighth embodiments and the samples prepared in the first to seventh comparative examples were selected, placed at room temperature for 7 days, and then applied to the skin membrane with a complete stratum corneum. After 24 hours, the receiving liquid was regularly extracted from the receiving pool under the skin membrane for analysis, and the sodium hyaluronate content in the receiving pool was tested by the glucuronic acid method. The test results are shown in Table 5.

[0050] The calculation method of transdermal absorption rate is as follows:

[0051] Where: C0—sodium hyaluronate content in the sample pool; C1—Sodium hyaluronate content when completely absorbed through the skin.

[0052] Table 5

[0053] As can be seen from Table 5, the aqueous solution hyaluronic acid oligosaccharide compositions prepared by the first to eighth embodiments have a higher transdermal rate, and are also improved to a certain extent compared with 0.8 kDa and 10 kDa ordinary hyaluronic acids; while the samples prepared by the first to seventh comparative examples have a lower transdermal rate, are not easily absorbed by the skin, and have limited anti-aging effects. This is mainly because with the cross-linking of the thiol groups, the molecular weight of the hyaluronic acid gradually increases, which affects its transdermal absorbability.

[0054] Fifth test case: Antioxidant effect test on zebrafish embryos 1. Test principle: CellROX ® The reagent is a DNA dye with weak fluorescence. It can be oxidized by oxygen free radicals (ROS) in cells. The oxidation products bind to DNA to produce bright green fluorescence. ® Soluble in organic solvents (such as dimethyl sulfoxide), and the main component of zebrafish yolk sac is fat, so CellROX ®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.

[0055] 2. Test method: 1) Group setting: Zebrafish were randomly selected in 6-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, and two wells (30 fish in total) were selected as the sample group.

[0056] 2) Feeding method: The model control group and the sample group were given menadione to establish a zebrafish oxidative stress model, wherein the sample group was given the aqueous solution hyaluronic acid oligosaccharide composition of the present invention (the sample prepared in the first embodiment was taken and diluted with distilled water to a total hyaluronic acid content of 0.5 mg / mL), 3 mL per well, while the model control group was not given the composition of the present invention and was fed normally; the normal control group was not given menadione, the zebrafish oxidative stress model was not established, and the composition of the present invention was not given, and was fed normally. The feeding conditions were incubated at 28 ° C in the dark for 22 h, and the zebrafish were stained with a specific ROS fluorescent reagent. After the staining, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photography, and the image processing software was used to analyze and collect data, and the zebrafish yolk sac fluorescence intensity (S) was analyzed, and the formula was used to calculate and determine whether the sample had an antioxidant effect.

[0057]

[0058] The experimental results are as follows Figure 1 As shown in the figure, it can be seen from the experimental results that the fluorescence intensity of the zebrafish yolk sac in the sample group is significantly reduced compared with the model control group, revealing that the sample has an antioxidant effect. According to the above calculation formula, the sample group has a zebrafish embryo reactive oxygen species (ROS) clearance rate of 73% (P < 0.001), which means that the composition prepared in the example has an antioxidant effect.

[0059] The sixth test case: anti-aging effect test of zebrafish embryos 1. Test principle: The aging process of zebrafish has certain similarities with that of humans. Hydrogen peroxide induces DNA damage response through oxidative stress and activates the p53 / p21 signaling pathway, leading to zebrafish aging. During oxidative stress, ROS in zebrafish increases, superoxide dismutase (SOD) activity decreases, and excessive lipid peroxidation product malondialdehyde (MDA) is produced. Catalase (CAT) and glutathione peroxidase (GSH-Px) are important peroxide decomposition enzymes in the body. Glutathione peroxidase (GSH-Px) catalyzes the oxidation of reduced glutathione (GSH) to generate oxidized glutathione (GSSG), thereby protecting the integrity of cell membranes and protecting cells from oxidative damage. Therefore, the anti-aging effect of the sample can be evaluated by detecting SOD activity.

[0060] 2. Test method: 1) Group setting: Zebrafish were randomly selected in 6-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, and two wells (30 fish in total) were selected as the sample group.

[0061] 2) Feeding method: The model control group and the sample group were given hydrogen peroxide to establish a zebrafish aging model, wherein the sample group was given the composition of the present invention (the sample prepared in the first embodiment was taken and diluted with distilled water to a total hyaluronic acid content of 0.5 mg / mL), 3 mL per well, while the model control group was not given the composition of the present invention and was fed normally; the normal control group was not given hydrogen peroxide, the zebrafish aging model was not established, and the composition of the present invention was not given, and was fed normally. The feeding conditions were incubated in the dark at 28 ° C for 120 h, and the liquid in the well was replaced once every 24 hours, with three biological replicates. After the experiment, the zebrafish samples of each group were collected, ground and homogenized, and the supernatant was taken by centrifugation. The SOD activity detection kit was used to collect data using an ELISA instrument to analyze the SOD activity (A) in the zebrafish, and the formula was used to calculate and determine whether the sample had an anti-aging effect.

[0062]

[0063] The experimental results are as follows Figure 2 As shown, it can be seen from the experimental results that the SOD activity of the sample group is significantly enhanced compared with the model control group, revealing that the sample has an anti-aging effect. According to the above clearance calculation formula, the dilution of the sample prepared in the first embodiment has an anti-aging effect on zebrafish embryos that is increased by 45% (P < 0.001), which means that the composition prepared in the embodiment has an anti-aging effect.

[0064] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an aqueous solution hyaluronic acid oligosaccharide composition, characterized in that: The steps include: Step 1, dissolving a hyaluronic acid raw material in water, then adding hydrochloric acid for acid hydrolysis to obtain an acid hydrolyzate, and then performing thiol modification on the hyaluronic acid in the acid hydrolyzate to obtain a thiol-modified hyaluronic acid solution; Step 2, dissolving the hyaluronic acid raw material in water, and then adding hydrochloric acid for acid hydrolysis to obtain the same acid hydrolysis solution as step 1; Step 3, mixing the thiolated hyaluronic acid solution obtained in step 1 and the acid hydrolyzate obtained in step 2 at a volume ratio of less than 1:5 to obtain a mixed solution; Step 4, placing the mixed solution obtained in step 3 into a dialysis bag with a molecular weight cutoff of 1.2 kDa for dialysis, and collecting the feed liquid in the dialysis bag; Step 5: Pass the liquid obtained in step 4 through an ultrafiltration membrane with a molecular weight cutoff of 4.0 kDa, collect the permeate, and obtain an aqueous solution hyaluronic acid oligosaccharide composition, wherein the total hyaluronic acid content is 0.2 mg / mL to 30 mg / mL, and the thiol content is 1 μmol / g polymer to 250 μmol / g polymer.

2. The preparation method according to claim 1, characterized in that In step 1 and step 2, the molecular weight of the hyaluronic acid raw material is 4 kDa to 200 kDa; The hyaluronic acid raw material is selected from hyaluronic acid or its salt.

3. The preparation method according to claim 2, characterized in that: The molecular weight of the hyaluronic acid raw material is 10kDa to 50kDa; The hyaluronic acid raw material is sodium hyaluronate.

4. The preparation method according to claim 1, characterized in that: In step 1, the thiol content is 5 μmol / g polymer to 100 μmol / g polymer.

5. The preparation method according to claim 1, characterized in that: In step 1 and step 2, the concentration of hydrochloric acid is 4 mol / L to 8 mol / L; The volume of hydrochloric acid is 2% to 4% of the volume of the hyaluronic acid solution; The acid hydrolysis time is 10 to 120 minutes.

6. The preparation method according to claim 5, characterized in that: The concentration of hydrochloric acid is 6 mol / L; The volume of hydrochloric acid is 3% of the volume of the hyaluronic acid solution.

7. The preparation method according to claim 1, characterized in that: In step 3, the volume ratio of the thiolated hyaluronic acid solution to the acid-hydrolyzed hyaluronic acid solution is 1:6 to 1:

49.

8. An aqueous solution hyaluronic acid oligosaccharide composition obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the aqueous solution hyaluronic acid oligosaccharide composition according to claim 8 in the preparation of anti-aging products.

10. The use according to claim 9, characterized in that The addition amount of the aqueous solution hyaluronic acid oligosaccharide composition is 0.2% to 100%.

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