Preparation method of sodium hyaluronate oligosaccharide composition, and composition and application prepared by the method

Abstract

The application belongs to the field of biochemistry, and particularly relates to a preparation method of a sodium hyaluronate oligosaccharide composition, and further discloses the prepared composition and application. The preparation method of the sodium hyaluronate oligosaccharide comprises the following steps: taking macromolecular sodium hyaluronate as raw material, preparing an acidic aqueous solution, adding ethanol to perform a precipitation reaction, separating supernatant, removing impurities through an ultrafiltration membrane, performing a second ethanol precipitation reaction, separating precipitate, purifying the precipitate through a reverse-phase ODS column, and obtaining the sodium hyaluronate oligosaccharide with required performance. The preparation method has the advantages of simple and easy-to-operate preparation process, suitable reaction conditions, simple and smooth production process, greatly reduced production period and cost, and a product yield of more than 96%. The sodium hyaluronate oligosaccharide composition prepared by the method comprises disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, decasaccharide and dodecasaccharide, has better product stability, excellent water solubility, good transdermal permeability, good moisturizing property, good oral absorption and other advantages.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry, specifically relating to a method for preparing a sodium hyaluronate oligosaccharide composition, and further disclosing the prepared composition and its application. Background Technology

[0002] Sodium hyaluronate, with the chemical formula (C14H20N011Na)n, is an inherent component of the human body. It is not species-specific and is widely found in tissues such as the placenta, amniotic fluid, lens, articular cartilage, and dermis, as well as in the cytoplasm and intercellular matrix of organs, playing a lubricating and nourishing role for the cells and organelles within them. Furthermore, sodium hyaluronate is a major component of the extracellular matrix and an important component of skin, vitreous humor, synovial fluid, and cartilage tissue. It possesses unique physicochemical properties and biological functions, playing a crucial role in maintaining tissue integrity and in tissue formation and remodeling during infection, injury, and embryonic development.

[0003] Sodium hyaluronate is classified according to its molecular weight, typically including: high molecular weight sodium hyaluronate (molecular weight ≥ 1.6 million Da); medium molecular weight sodium hyaluronate (molecular weight 200,000-1.6 million Da); low molecular weight sodium hyaluronate (molecular weight 10,000-200,000 Da); and low molecular weight sodium hyaluronate (molecular weight 3,000 Da-10,000 Da). Although sodium hyaluronate is widely used in medical and pharmaceutical fields, daily chemical products, nutritional foods, and pet food, the currently used sodium hyaluronate generally has a large molecular weight and is too viscous, resulting in poor transdermal absorption, poor oral absorption, and poor bioavailability, thus limiting its application scenarios. Therefore, the development of low molecular weight sodium hyaluronate and even sodium hyaluronate oligosaccharides has been a focus of attention for researchers in this field.

[0004] In existing technologies, the preparation of low molecular weight sodium hyaluronate products is mostly based on methods that degrade high molecular weight sodium hyaluronate, mainly including physical methods, chemical methods, and enzymatic hydrolysis. However, these methods generally suffer from problems such as large molecular weight of degradation products, wide molecular weight range of products, unstable degradation effects, and high costs. For example, the method for preparing low molecular weight sodium hyaluronate disclosed in Chinese patent CN101020724A can control the average molecular weight of the obtained sodium hyaluronate between 8000-20000 Da, with a minimum of about 5000 Da. The wide molecular weight range of the sodium hyaluronate prepared by this method cannot meet market demands. The method for producing low molecular weight hyaluronic acid using hydrogen peroxide and ascorbic acid oxidative degradation disclosed in Chinese patent CN101293934A has problems such as residual organic solvents, complex post-processing, and environmental damage. For example, Chinese patents CN103484513A and CN101507733A disclose methods for preparing small-molecule oligomeric hyaluronic acid using enzymatic methods. However, these methods utilize gene recombination technology, making the process extremely complex and costly. In summary, existing sodium hyaluronate products typically suffer from unstable quality and significant product variations, such as excessively large molecular weight distribution, uneven color, low solubility, and prolonged dissolution time. Furthermore, sodium hyaluronate preparation methods generally suffer from low efficiency (around 50%, rarely exceeding 90%), high costs, and residual organic solvents. Invention Overview

[0006] Technical issues

[0007] Therefore, the technical problem to be solved by the present invention is to provide a method for preparing a sodium hyaluronate oligosaccharide composition, which has the advantages of simple process operation, low production cost and high production efficiency.

[0008] The second technical problem to be solved by the present invention is to provide a sodium hyaluronate oligosaccharide composition product with stable product quality, high solubility and rapid dissolution prepared by the above preparation method; the sodium hyaluronate oligosaccharide composition product can not only effectively improve the transdermal absorption effect, but also effectively increase the skin moisture content and have good moisturizing properties;

[0009] The third technical problem to be solved by the present invention is to provide the use of the above-mentioned sodium hyaluronate oligosaccharide composition.

[0010] Solution to the problem

[0011] Technical solutions

[0012] To solve the above-mentioned technical problems, the present invention provides a method for preparing the sodium hyaluronate oligosaccharide composition, comprising the following steps:

[0013] (1) Take sodium hyaluronate as a raw material to prepare an acidic aqueous solution and carry out the reaction;

[0014] (2) Adjust the pH of the above reaction solution to neutral, add ethanol and make the ethanol concentration reach 50-70%, and collect the first precipitate and the supernatant separately for later use;

[0015] (3) Remove impurities from the supernatant by ultrafiltration, add ethanol to make the ethanol concentration reach 70-90%, and collect the precipitated second precipitate;

[0016] (4) Dissolve the second precipitate in water and purify it using a reversed-phase ODS column to obtain the final product.

[0017] Specifically, the preferred ODS column has a filler particle size of 20-50 μm.

[0018] Specifically, in step (1), the molecular weight of the macromolecular sodium hyaluronate is 50,000 to 2,000,000 Da;

[0019] The acid used to prepare the acidic aqueous solution is sulfuric acid, hydrochloric acid, hydrofluoric acid or nitric acid, preferably sulfuric acid and hydrochloric acid, and particularly preferably sulfuric acid;

[0020] The concentration of the acid is 0.5-4.0M; more preferably 0.5M-1.5M, and particularly preferably 0.8-1.2M;

[0021] The mass ratio of the macromolecular sodium hyaluronate to the acid is 1:5-15; the reaction temperature is 50-150℃, and the reaction time is 3-15 hours. More preferably, the temperature is 60-120℃, and particularly preferably 70-80℃; the reaction time is further preferably 6-8 hours.

[0022] Specifically, in step (2), the alkali used to adjust the pH of the reaction solution includes sodium hydroxide, potassium hydroxide, sodium carbonate, etc., preferably sodium hydroxide.

[0023] Specifically, in step (3), the molecular weight cutoff of the ultrafiltration membrane is 1500-3000 da.

[0024] Specifically, in step (4), the purification step includes elution with 0.5-3 v / v% ethanol aqueous solution and elution with 40-60 v / v% ethanol aqueous solution. The elution fraction of 40-60 v / v% ethanol is collected, the ethanol is recovered and dried to obtain the final product.

[0025] Preferably, in step (4), the purification step includes eluting 1-4 column volumes with 1 v / v% ethanol aqueous solution and eluting 2-4 column volumes with 50 v / v% ethanol aqueous solution, collecting the 50 v / v% ethanol eluent, recovering the ethanol and drying it to obtain the final product.

[0026] Specifically, the method further includes the steps of dissolving the first precipitate in water and purifying it with an activated carbon column, collecting the purified product and repeating steps (1)-(2); the number of times steps (1)-(2) are repeated is 1-3 times.

[0027] Specifically, the purification step using an activated carbon column includes elution with water and elution with a 20-40 v / v% ethanol aqueous solution. The elution fraction of the 20-40 v / v% ethanol aqueous solution is collected, the ethanol is recovered, and the product is dried to obtain the desired purified product.

[0028] Preferably, the purification step using an activated carbon column includes eluting with water for 4-6 column volumes, and eluting with a 30 v / v% ethanol aqueous solution, collecting the eluent from the 30 v / v% ethanol aqueous solution, recovering the ethanol, and drying to obtain the desired purified product.

[0029] Specifically, the preparation method of the sodium hyaluronate oligosaccharide is as follows:

[0030] The second precipitate was dissolved in water to a concentration of 10-25 mg / mL.

[0031] The first precipitate was dissolved in water to a concentration of 20-35 mg / mL.

[0032] In the preparation method of the sodium hyaluronate oligosaccharide composition of the present invention, the main purpose of adjusting the pH is to enable hyaluronic acid to form sodium hyaluronate salt under acidic conditions. In acidic solution, the raw material sodium hyaluronate will be acidified into hyaluronic acid. Here, the pH is adjusted to neutral to neutralize the acidity in the solution, allowing hyaluronic acid to form sodium hyaluronate salt, which is more soluble in water and more likely to precipitate when ethanol is added.

[0033] The research of this invention found that the final concentration of ethanol is 70-90%, which is optimal. When the concentration is lower than 70%, the precipitated sodium hyaluronate is viscous and doughy, which makes subsequent processing very troublesome. When the concentration is higher than 90%, the amount of impurities will increase, and the cost will also increase.

[0034] This invention uses an activated carbon column to desalt and decolorize the first precipitate. The activated carbon column can adsorb sodium hyaluronate of various molecular weights. Washing with water removes the salt from the first precipitate, and then eluting the sodium hyaluronate with an ethanol-water solution, while the pigment remains on the activated carbon column. Activated carbon is low in cost and not only effectively desalts but also decolorizes, thus effectively purifying sodium hyaluronate.

[0035] ODS (Optical Dispersant Analyzer) is originally a chromatographic packing material used for purifying highly polar substances. This invention creatively applies it to the purification and desalting of sodium hyaluronate oligosaccharides, employing specific purification and desalting conditions, including initial elution with a low-concentration ethanol-water solution followed by elution of the hyaluronate oligosaccharides with a high-concentration ethanol-water solution. This invention has found that using ODS to purify sodium hyaluronate oligosaccharides not only provides excellent desalting and high purification efficiency but also yields a high amount of hyaluronate oligosaccharides. After a single ODS treatment, the purity of sodium hyaluronate oligosaccharides can reach over 96%, a significant advantage compared to other purification methods that require multiple purification processes to achieve the desired purity.

[0036] The present invention also discloses a sodium hyaluronate oligosaccharide composition prepared by the above method, wherein the general molecular formula of the sodium hyaluronate oligosaccharide is (C14H20N011Na)1-6, including: sodium hyaluronate disaccharide, sodium hyaluronate tetrasaccharide, sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, sodium hyaluronate decasaccharide and sodium hyaluronate dodecasaccharide.

[0037] Wherein, based on the total amount of the oligosaccharide composition being 100%, the sum of the mass contents of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide and sodium hyaluronate decasaccharide is 40-60 wt%.

[0038] Preferably, the mass content of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, and sodium hyaluronate decasaccharide is 45%-60%, more preferably 45%-55%, and even more preferably 50%-55%.

[0039] Specifically, based on the total amount of the oligosaccharide composition as 100%, the content of sodium hyaluronate disaccharide is 10-24%, the content of sodium hyaluronate tetrasaccharide is 20%-33%, and the content of sodium hyaluronate dodecanose is 3%-8%.

[0040] Specifically, the mass ratio of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, and sodium hyaluronate decasaccharide is 20-26:13-19:7-17;

[0041] Specifically, the average molecular weight of the sodium hyaluronate oligosaccharide is 1100-1600 Da, and the pH value of a 1 wt% aqueous solution is 6-7;

[0042] According to the colorimeter method in the Chinese Pharmacopoeia, the L* value of the sodium hyaluronate oligosaccharide composition product is 95-99.99, the a* value is -0.2-0.2, the b* value is -0.2-0.2, the ΔL value is 18-21, the Δa value is 36-39, the Δb value is 32-35, and the ΔE value is 93-96.

[0043] The sodium hyaluronate oligosaccharide composition product has a maximum absorption peak at 190 nm in the ultraviolet spectrum, a maximum solubility of 25 g / 100 mL in water, and the solution is a clear and transparent solution. The optical rotation at a concentration of 1 wt% is -0.7 to 0.9, the ash content is 12% to 15%, and the aqueous solution can pass through an ultrafiltration membrane with a rejection value of 3000 Da, achieving a dry matter conversion rate of 100%.

[0044] The present invention also discloses the use of the sodium hyaluronate oligosaccharide composition or the sodium hyaluronate oligosaccharide composition prepared by the method for the preparation of food, medical products and / or daily chemical products.

[0045] Specifically, the food includes solid food, semi-solid food, and liquid food.

[0046] The sodium hyaluronate oligosaccharide described in this invention, when used in oral foods, has cosmetic and joint health-improving effects. It can improve skin hydration, provide antioxidant benefits, enhance skin elasticity, reduce joint inflammation, and improve overall joint health. Therefore, it can be widely used in powders, granules, soft capsules, capsules, tablets, solid beverages, liquid beverages, dairy products, and other products.

[0047] Specifically, the food may include pet food, specifically solid, semi-solid, and liquid pet food. The sodium hyaluronate oligosaccharide of this invention, when used in pet food, effectively supplements the deficiency of sodium hyaluronate in dogs and cats through oral administration, providing multiple benefits such as nourishing the skin and promoting shiny fur, improving joint health, and protecting the digestive system. Therefore, it can be used in the development and application of various pet foods, including dog and cat staple foods and health supplements.

[0048] Specifically, the medical supplies may include conventional pharmaceuticals in the art; more specifically, they may include eye drops, lubricating eye drops, contact lens care solutions, eye washes, cavity lubricants, oral medications, adjuvant medications for ophthalmic surgery, medications for improving knee arthritis, osteoarthritis, and frozen shoulder, dermal fillers, anti-adhesion preparations, etc.

[0049] Specifically, the daily chemical products include regular daily necessities and cosmetics.

[0050] Specifically, the cosmetics can include product forms commonly used in the art, including masks, serums, gels, facial cleansers, toothpaste, mouthwash, scalp care products, sunscreens, eye creams, cushion foundations, sprays, freeze-dried powders, etc. The sodium hyaluronate oligosaccharide described in this invention, when used in cosmetics, can be instantly absorbed through the skin, reaching the dermis to moisturize, repair damaged cells, relieve skin sensitivity, reduce inflammation, scavenge free radicals, nourish epidermal cells, improve skin elasticity, reduce wrinkles, rebuild a healthy skin barrier, and exert anti-aging effects. Therefore, it can be widely used in anti-aging, sun protection, moisturizing, soothing, redness reduction, repair, and acne treatment cosmetics.

[0051] The sodium hyaluronate oligosaccharide described in this invention can be used in oral care, and has multiple effects such as moisturizing, lubrication, anti-inflammation, and repair. It can be used in the development and application of various oral cleaning and care products such as mouthwash, spray, and gel.

[0052] The sodium hyaluronate oligosaccharide described in this invention is used in the field of family planning and has advantages such as good compatibility with natural latex, complete water solubility, easy washing, long-lasting lubrication, non-stickiness, and moisturizing and nourishing properties. The sodium hyaluronate oligosaccharide lubricant can be mixed with other water-soluble ingredients or directly soaked in condoms to reduce mucosal damage and nourish delicate skin.

[0053] Beneficial effects of the invention

[0054] Beneficial effects

[0055] The method for preparing sodium hyaluronate oligosaccharides according to this invention involves using large-molecule sodium hyaluronate as raw material, preparing an acidic aqueous solution, and then adding ethanol of a selected concentration to induce a precipitation reaction. The supernatant is separated, purified by ultrafiltration, and subjected to a second precipitation reaction with ethanol of a selected concentration. The precipitate is then purified by reverse-phase ODS column chromatography to obtain sodium hyaluronate oligosaccharides with the desired properties. The method described in this invention is simple and easy to implement, with suitable reaction conditions and a streamlined production process, significantly reducing the production cycle and cost. The product yield can reach over 96%, offering advantages such as ease of operation and low cost. It effectively overcomes the shortcomings of traditional enzymatic hydrolysis methods, which are complex and costly, and traditional physical methods, which are relatively complex and require sophisticated equipment. This method is suitable for large-scale industrial production of sodium hyaluronate oligosaccharides.

[0056] The sodium hyaluronate oligosaccharide composition prepared by the method of the present invention comprises oligosaccharides with the general molecular formula (C14H20NO11Na)1-6, and its systematic name is: [(1→4)-O-β-D-glucuronic acid-(1→3)-2-acetamido-2-deoxy-β-D-glucose]1-6 sodium salt, including disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, and dodecasaccharides. The method of the present invention, through adjustment of the process and control parameters, yields a sodium hyaluronate oligosaccharide composition product with a well-defined structure and molecular weight, exhibiting better product stability, excellent water solubility, good transdermal permeability, moisturizing properties, and good oral absorption than ordinary sodium hyaluronate, making it suitable for use in the food, medical, and cosmetic fields.

[0057] The sodium hyaluronate oligosaccharide composition product prepared by the method of the present invention has a total mass content of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, and sodium hyaluronate decasaccharide of more than 40%. It not only has good transdermal absorption performance, but also, because its molecular weight is larger than that of sodium hyaluronate disaccharide and sodium hyaluronate tetrasaccharide, its metabolism in the skin is relatively slower, which can better retain the moisture in the skin. Compared with other sodium hyaluronate oligosaccharide products, it can not only effectively improve the transdermal absorption effect, but also effectively increase the skin moisture content and have good moisturizing properties.

[0058] Brief description of the accompanying drawings Attached Figure Description

[0059] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0060] Figure 1 The gel chromatogram of the sodium hyaluronate oligosaccharide sample described in Experimental Example 1;

[0061] Figure 2 The results of skin moisture detection in rats in Experiment Example 2 are compared.

[0062] Figure 3 The curves showing the changes in absorbance of the receiving liquid at different transdermal times after treatment in Experiment Example 3 are shown.

[0063] Figure 4 This is a comparison chart of the moisturizing abilities of different moisturizers in Experiment Example 4.

[0064] Invention Embodiments

[0065] Embodiments of the present invention

[0066] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0067] In the following embodiments of the present invention, the purity of the sodium hyaluronate oligosaccharide was determined by ultraviolet spectrophotometry according to QB / T4416-201 "Cosmetic Raw Materials - Sodium Hyaluronate".

[0068] Example 1

[0069] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment includes the following steps:

[0070] (1) Take 500g of large molecular weight sodium hyaluronate (average molecular weight 1.2 million Da) and place it in a 20L reactor. Add 1M sulfuric acid. The mass ratio of large molecular weight sodium hyaluronate to sulfuric acid is 1:10. Turn on the circulating water heating pump and heat to 70℃. Start timing the reaction and keep the temperature constant for 7 hours for hydrolysis.

[0071] (2) Add sodium hydroxide aqueous solution to the above reaction solution under stirring to adjust the pH to 7, add an appropriate amount of ethanol to make its concentration reach 50%, and carry out the reaction until no precipitate is produced. Collect the first precipitate and the supernatant separately.

[0072] The first precipitate was dissolved in distilled water to a concentration of 30 mg / mL as a sample. The sample was purified using an activated carbon column. After eluting with water for 5 column volumes, the sample was eluted with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C. The purified product was obtained by freeze-drying.

[0073] The purified product was used as a raw material to repeat steps (1)-(2) twice.

[0074] (3) After combining the above supernatants and removing impurities through an ultrafiltration membrane, add ethanol to make its concentration reach 70%, and react until no precipitate is produced. Collect the precipitated second precipitate.

[0075] (4) The second precipitate was dissolved in distilled water to a concentration of 20 mg / mL, purified by reverse-phase ODS column (particle size 20 μm), eluted with 1 v / v% ethanol-water solution for 2 column volumes, and then eluted with 50 v / v% ethanol for 4 column volumes. The 50 v / v% ethanol eluent was collected, and after ethanol recovery under reduced pressure, it was freeze-dried to obtain 495.5 g of the required sodium hyaluronate oligosaccharide. The yield was calculated to be 99.1% and the purity was 98.8%.

[0076] Example 2

[0077] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment includes the following steps:

[0078] (1) Take 500g of large molecular weight sodium hyaluronate (average molecular weight 600,000 Da) and place it in a 20L reactor. Add 1M hydrochloric acid. The mass ratio of large molecular weight sodium hyaluronate to hydrochloric acid is 1:10. Turn on the circulating water heating pump and heat to 80℃. Start timing the reaction and keep the temperature constant for 8 hours for hydrolysis.

[0079] (2) Add sodium hydroxide aqueous solution to the above reaction solution under stirring to adjust the pH to 7, add an appropriate amount of ethanol to make its concentration reach 70%, and carry out the reaction until no precipitate is produced. Collect the first precipitate and the supernatant separately.

[0080] The first precipitate was dissolved in distilled water to a concentration of 25 mg / mL as a sample. The sample was purified using an activated carbon column. After eluting with water for 5 column volumes, the sample was eluted with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C. The purified product was obtained by freeze-drying.

[0081] The purified product was used as a raw material to repeat steps (1)-(2) twice.

[0082] (3) After combining the above supernatants and removing impurities through an ultrafiltration membrane, add ethanol to make its concentration reach 90%, and react until no precipitate is produced. Collect the precipitated second precipitate.

[0083] (4) The second precipitate was dissolved in distilled water to a concentration of 20 mg / mL, purified by reverse-phase ODS column (particle size 20 μm), eluted with 1 v / v% ethanol-water solution for 2 column volumes, and then eluted with 50 v / v% ethanol for 4 column volumes. The 50 v / v% ethanol eluent was collected, and after the ethanol was recovered under reduced pressure, it was freeze-dried to obtain 485.5 g of the required sodium hyaluronate oligosaccharide. The yield was calculated to be 97.1% and the purity was 97.6%.

[0084] Example 3

[0085] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment includes the following steps:

[0086] (1) Take 1 kg of large molecular weight sodium hyaluronate (average molecular weight 1.2 million Da) and place it in a 100L enamel reactor. Add 1M sulfuric acid. The mass ratio of large molecular weight sodium hyaluronate to sulfuric acid is 1:10. Stir to dissolve and heat to 70℃. Start timing the reaction and keep the temperature constant for 7 hours for hydrolysis.

[0087] (2) Add sodium hydroxide aqueous solution to the above reaction solution under stirring to adjust the pH to 7, and slowly add ethanol under stirring until the ethanol concentration is 60%. Continue the reaction until no precipitate is formed, and collect the first precipitate and the supernatant separately.

[0088] The first precipitate was dissolved in distilled water to a concentration of 35 mg / mL as a sample. The sample was purified using an activated carbon column. After eluting with water for 4 column volumes, the sample was eluted with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C. The purified product was then freeze-dried.

[0089] The purified product was used as a raw material to repeat steps (1)-(2) twice.

[0090] (3) After combining the above supernatants and removing impurities through an ultrafiltration membrane, slowly add ethanol with stirring until the ethanol concentration is 80%, and react until no precipitate is formed. Collect the precipitated second precipitate.

[0091] (4) The second precipitate was dissolved in distilled water to a concentration of 25 mg / mL, purified by reverse-phase ODS column (particle size 50 μm), eluted with 1 v / v% ethanol-water solution for 2 column volumes, and then eluted with 50 v / v% ethanol for 2 column volumes. The 50 v / v% ethanol eluent was collected, and after ethanol recovery under reduced pressure, it was freeze-dried to obtain 968.6 g of the required sodium hyaluronate oligosaccharide. The yield was calculated to be 96.9% and the purity was 98.2%.

[0092] Example 4

[0093] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment includes the following steps:

[0094] (1) Take 20 kg of large molecular weight sodium hyaluronate (average molecular weight 1.2 million) and put it into a 1-ton enamel reactor. Add 1M sulfuric acid. The mass ratio of large molecular weight sodium hyaluronate to sulfuric acid is 1:10. Stir to dissolve and heat to 70°C. Start timing the reaction and keep the temperature constant for 7 hours for hydrolysis.

[0095] (2) Add sodium hydroxide aqueous solution to the above reaction solution under stirring to adjust the pH to 7, and slowly add ethanol under stirring until the ethanol concentration is 60%. Continue the reaction until no precipitate is formed, and collect the first precipitate and the supernatant separately.

[0096] The first precipitate was dissolved in distilled water to a concentration of 35 mg / mL as a sample. The sample was purified using an activated carbon column. After eluting with water for 4 column volumes, the sample was eluted with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C. The purified product was then freeze-dried.

[0097] The purified product was used as a raw material to repeat steps (1)-(2) twice.

[0098] (3) After combining the above supernatants and removing impurities through an ultrafiltration membrane, slowly add ethanol with stirring until the ethanol concentration is 80%, and react until no precipitate is formed. Collect the precipitated second precipitate.

[0099] (4) The second precipitate was dissolved in distilled water to a concentration of 25 mg / mL and purified by reversed-phase ODS column (particle size 50 μm). It was eluted with 1 v / v% ethanol-water solution for 1.5 column volumes, and then eluted with 50 v / v% ethanol for 2 column volumes. The 50 v / v% ethanol eluent was collected, and after the ethanol was recovered under reduced pressure, it was freeze-dried to obtain 19.3 kg of the required sodium hyaluronate oligosaccharide. The yield was calculated to be 96.5% and the purity was 96.3%.

[0100] Comparative Example 1

[0101] The preparation method of the sodium hyaluronate oligosaccharide composition described in this comparative example is the same as that in Example 4, except that the hydrolysis reaction time in step (1) is extended to 24 hours.

[0102] Comparative Example 2

[0103] The preparation method of the sodium hyaluronate oligosaccharide composition described in this comparative example is the same as that in Example 4, except that in step (3), the molecular weight cutoff of the ultrafiltration membrane is 1000 Da.

[0104] Experimental Example

[0105] Experimental Example 1: Performance Testing of Sodium Hyaluronate Oligosaccharide Composition Products

[0106] The sodium hyaluronate oligosaccharide composition prepared in Example 4 was used as a sample for the following tests.

[0107] Molecular weight and molecular weight distribution detection

[0108] The determination was performed according to the size exclusion chromatography method in Chapter 0514 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition). The gel chromatogram of the sodium hyaluronate oligosaccharide sample (Example 4) is attached. Figure 1 As shown in the table below, the molecular weight and molecular weight distribution of the sodium hyaluronate oligosaccharide were determined.

[0109] Molecular weight and molecular weight distribution of sodium hyaluronate oligosaccharides

[0110] [Table 1]

[0111] project Test results Mw <![CDATA[1.275×10 3 g / mol]]> Mn <![CDATA[1.162×10 3 g / mol]]> Mw / Mn 1.098

[0112] Filter membrane detection

[0113] The sodium hyaluronate oligosaccharide sample of the present invention (Example 4) was tested using a 3000 molecular weight filter membrane. The result showed that the dry matter yield was 100%, indicating that all samples could pass through a 3000 Da filter membrane, meaning that the maximum molecular weight of the obtained sodium hyaluronate oligosaccharide did not exceed 3000 Da.

[0114] Solubility test

[0115] The sodium hyaluronate oligosaccharide sample (Example 4) of this invention has a solubility of 2.5g dissolved in 10mL of water. When the amount exceeds 2.5g, a precipitate is formed, indicating that the solubility of the obtained sodium hyaluronate oligosaccharide is 25g / 100mL.

[0116] The sodium hyaluronate oligosaccharide compositions prepared in Examples 1-4 and Comparative Examples 1-2 were used as samples for HPLC analysis. The HPLC analysis was performed according to the high-performance liquid chromatography method (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 0512), using 20% ​​acetonitrile-water as the mobile phase; flow rate 0.8 mL / min; detection: ultraviolet light, wavelength 191 nm; column temperature: 30℃. It should be noted that HPLC analysis can only determine the content of each hyaluronic acid oligosaccharide component in the sample; salt impurities such as Na₂SO₄ and NaCl cannot be detected.

[0117] According to HPLC results, the sodium hyaluronate oligosaccharide composition prepared in this invention includes sodium hyaluronate disaccharide, sodium hyaluronate tetrasaccharide, sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, sodium hyaluronate decasaccharide, and sodium hyaluronate dodecasaccharide. The general molecular formula of sodium hyaluronate oligosaccharides is: (C 14 H 20 NO 11 Na) 1-6 Its systematic name is: [(1→4)-O-β-D-glucuronic acid-(1→3)-2-acetamido-2-deoxy-β-D-glucose] 1-6 Sodium salt.

[0118] The mass content of each component in the sodium hyaluronate oligosaccharide composition sample is shown in Table 1 below, where Rt refers to the retention time.

[0119] Table 1. Sample content of sodium hyaluronate oligosaccharides

[0120]

[0121] Experiment Example 2, Animal Experiment

[0122] This experiment was conducted to observe the changes in rats after feeding them a diet containing sodium hyaluronate oligosaccharide.

[0123] Experimental materials

[0124] Sodium hyaluronate oligosaccharide sample (Example 4), sodium hyaluronate oligosaccharide control group (Comparative Example 1); experimental rat food, produced by an animal food factory. Based on the rat food, specially formulated rat food containing different amounts of sodium hyaluronate oligosaccharide was prepared (the original rat food was pulverized, and 0.02%, 0.06%, and 0.09% (mass content) of sodium hyaluronate oligosaccharide were added to the rat food, respectively, and then the resulting experimental rat food pellets with different sodium hyaluronate oligosaccharide contents were made).

[0125] Experimental testing instruments

[0126] The Corneometer CM 825 skin moisture meter is manufactured by the German company Courage+Khazaka.

[0127] laboratory animals

[0128] All experimental rats were SD strain clean-grade aged female rats, weighing 300-400g and 15 months old, provided by the Laboratory Animal Center of the Academy of Military Medical Sciences, China. Certificate No.: 035; Laboratory Animal Environment and Facility Certificate No.: 027. The animals were randomly divided into 7 groups of 20 each.

[0129] Experimental methods

[0130] One hundred and forty 15-month-old SD strain clean-grade rats, weighing (360±21)g, were randomly divided into seven groups of 20 rats each.

[0131] Three groups were divided into a normal feeding control group, three groups of diets containing sodium hyaluronate oligosaccharides, and three groups of diets containing sodium hyaluronate oligosaccharides (comparative example 1). The animals were fed the same diets containing low, medium, and high doses of sodium hyaluronate oligosaccharides for a total of 60 days. During this period, all experimental animals were housed individually in cages and had free access to food and water.

[0132] During the feeding period, the food intake, mental state, and activity of the rats in each group were observed daily. The presence of nausea and vomiting, as well as the odor and characteristics of their excrement (urine, feces, etc.), were also noted. Body weight was measured periodically. No rats died or were near death during the experiment.

[0133] Detection of skin water content in experimental rats

[0134] The tests were conducted in a spacious, well-ventilated room with relatively stable temperature (22℃±2℃) and humidity (60%). All rats were shaved along both sides of the spine on their backs, with a shaved area of ​​approximately 1.5cm × 1.5cm. The moisture content of the shaved area was measured using a skin moisture meter. All rat tests were performed on the same instrument by the same personnel in the same room. During testing, the contact force and vertical angle between the testing head and the rat's skin should be consistent.

[0135] Experimental results

[0136] The skin moisture measurements obtained in this experiment are summarized in Table 2 below. A comparison chart of rat skin moisture measurements is attached. Figure 2 .

[0137] Table 2 Summary and analysis of skin moisture detection data in experimental rats

[0138] [Table 2]

[0139]

[0140] As can be seen from the data in the table above, the skin water content of experimental animals increased synchronously with the increase in the amount of sodium hyaluronate oligosaccharide added to the feed.

[0141] The general condition of the rats was as follows: During the experiment, the rats in all groups grew well, and no obvious abnormalities were observed in their general condition, such as behavior, activity, mental state, water intake, and feces.

[0142] Data on skin moisture content showed no significant difference between the low-dose sodium hyaluronate oligosaccharide group and the blank control group; however, significant differences were observed between the medium- and high-dose sodium hyaluronate oligosaccharide groups and the blank control group, indicating that medium- and high-dose sodium hyaluronate oligosaccharides can significantly increase skin moisture content in rats. While the three doses in the sodium hyaluronate oligosaccharide control group showed an increase in skin moisture content compared to the blank control group, no significant difference was observed. In the sodium hyaluronate oligosaccharide composition of this invention, the content of sodium hyaluronate hexasaccharide to sodium hyaluronate decasaccharide is 40-60%, while the sodium hyaluronate oligosaccharide control group (Comparative Example 1) contains approximately 75% sodium hyaluronate disaccharide and less than 15% sodium hyaluronate hexasaccharide to sodium hyaluronate decasaccharide. Because sodium hyaluronate disaccharide has a small molecular weight, it is metabolized quickly after absorption and is easily lost, while sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, and sodium hyaluronate decasaccharide have large molecular weights, are metabolized slowly after absorption, and are less prone to loss, thus better maintaining skin moisture content.

[0143] It is evident that the sodium hyaluronate oligosaccharide composition product described in this invention has a good effect on increasing the skin moisture content of rats, and high skin moisture content indicates high skin health. Since animal skin also contains sodium hyaluronate, the maturation and aging process of rat skin varies with the content and metabolism of sodium hyaluronate. When the sodium hyaluronate content is high, it can improve skin nutrition and metabolism, making the skin soft, smooth, and promoting health. Similarly, sodium hyaluronate oligosaccharides, when used in pet food, can effectively supplement the deficiency of sodium hyaluronate in dogs and cats through oral administration, playing multiple roles such as nourishing the skin, smoothing the fur, improving joints, and protecting the gastrointestinal tract. Therefore, it can be used in the development and application of various pet foods such as dog and cat staple foods and health supplements.

[0144] Experimental Example 3 Transdermal Effect

[0145] This experiment uses ordinary sodium hyaluronate solution (200,000-400,000 Da), sodium hyaluronate oligosaccharide solution (sample of Example 4), and sodium hyaluronate oligosaccharide solutions of Comparative Examples 1-2 (as control groups 1-2) to conduct a comparative study to examine their transdermal effect on snake slough and provide certain experimental basis for their application.

[0146] Experimental instruments and reagents

[0147] Transdermal drug diffusion assay apparatus (Shanghai Youpu Scientific Instruments Co., Ltd.); UV-Vis spectrometer (Thermoelectric Corporation, USA); PBS solution was phosphate buffer solution, pH 7.5, prepared in-house.

[0148] Experimental methods

[0149] In vitro transdermal diffusion assay: Snake slough was purchased and soaked in physiological saline for 30 min, then stored in PBS solution at 4℃ for 12 h before use.

[0150] Cut a piece of snake skin of appropriate size and fix it in a transdermal drug diffusion tester (diffusion area 1.0 cm²). 2 The receiving pool has a volume of 8.5 cm³. 3 In the experiment, the outer surface of the snake slough faced the supply chamber, and the receiving chamber contained PBS buffer solution. The temperature was 37℃, and the mixture was stirred. Samples of 0.5 mL were taken at fixed intervals, and an equal volume of receiving solution was added after each sample collection. After the experiment, the sample solution was appropriately diluted, filtered through a 0.50 μm microporous membrane, hydrolyzed, and the absorbance at 550 nm was measured using ultraviolet spectrophotometry.

[0151] Experimental results

[0152] In vitro transdermal diffusion experiments were conducted, divided into three groups: the sodium hyaluronate oligosaccharide group (2 mL of a self-prepared 30 wt% sodium hyaluronate oligosaccharide solution), the control group (2 mL of a 30 wt% ordinary sodium hyaluronate solution), and the control group (30 wt% sodium hyaluronate oligosaccharide solutions from comparative examples 1-2). The absorbance results of the receiving solution at different transdermal times are shown in Table 3 below. The absorbance change curves of the receiving solution at different transdermal times after treatment are shown in the appendix. Figure 3 As shown.

[0153] Table 3. Absorbance of the receiving fluid at different transdermal times after treatment.

[0154]

[0155] As shown in the table above, the absorbance of the sodium hyaluronate oligosaccharide group was significantly higher than that of the ordinary sodium hyaluronate control group at the same transdermal time. This indicates that the permeability of the sodium hyaluronate oligosaccharide group through snake skin was significantly higher than that of the ordinary sodium hyaluronate control group at the same transdermal time, suggesting that the sodium hyaluronate oligosaccharide group with a molecular weight of less than 3000 can effectively improve the ability to penetrate snake slough. In control groups 1 and 2, due to the higher content of sodium hyaluronate disaccharide and sodium hyaluronate tetrasaccharide, the transdermal absorption effect was also better than that of the ordinary sodium hyaluronate group, but there was almost no difference compared with the sodium hyaluronate oligosaccharide group.

[0156] Experiment Example 4 Moisturizing Performance

[0157] This experiment compares the moisturizing properties of sodium hyaluronate with different molecular weights and commonly used moisturizing ingredients.

[0158] Experimental reagents and instruments

[0159] Sodium hyaluronate (200,000-400,000 Da), small molecule sodium hyaluronate (10,000-100,000 Da), cosmetic grade, Bloomage Freda Biotechnology Co., Ltd.

[0160] Sodium hyaluronate oligosaccharide, sample of Example 4; sodium hyaluronate oligosaccharide control group 1, sample of Comparative Example 1; sodium hyaluronate oligosaccharide control group 2, sample of Comparative Example 2;

[0161] Aloe vera gel extract, cosmetic grade, Beijing Sanyou Huizhi Biotechnology Co., Ltd.;

[0162] Tremella fuciformis extract, cosmetic grade, Huanya Research Institute;

[0163] Ceramide 3, cosmetic grade, Doosan Group, South Korea;

[0164] Hydrogenated polydecene, cetearyl alcohol, ARLACEL 165 (a mixture of glyceryl stearate and PEG-100 stearate), polydimethylsiloxane (5CST), carbomer, triethanolamine, xanthan gum, aminomethylpropanol, disodium EDTA and other excipients are all cosmetic grade and provided by cosmetic companies.

[0165] Corneometer CM825 skin moisture meter, Courage+Khazaka, Germany.

[0166] Experimental methods

[0167] Oil phase: hydrogenated polydecene 5.0, cetearyl alcohol 1.0, ARLACEL 165 3.0, polydimethylsiloxane 5.0;

[0168] Aqueous phase: EDTA disodium 0.02, carbomer 0.3, xanthan gum 0.1, triethanolamine 0.05, water balance;

[0169] Of the eight moisturizers, except for ceramide which is soluble in the oil phase, the other seven are soluble in the water phase. The dosages are shown in Table 4 below.

[0170] After the humectant is added to the oil or aqueous phase, the oil and aqueous phases are completely dissolved in an 80°C water bath. The oil phase is then slowly poured into the aqueous phase, and the mixture is pre-emulsified for 3 minutes at a stirring speed of 1000 rpm. The homogenizer is then turned on and the speed is increased to 9000 rpm for 3 minutes. After cooling to 50°C, aminomethylpropanol and preservatives are added, water is added, and the mixture is homogenized for another 1 minute. After the paste cools to room temperature, it is vacuum-discharged to obtain the final product.

[0171] Based on relevant literature and experimental experience, two levels of different moisturizers were selected for the experiment, and the specific addition amounts are shown in Table 4 below.

[0172] Table 4. Dosage of different moisturizers

[0173]

[0174] Forty-five healthy volunteers aged 20-50 years without allergic diseases were selected and divided into nine groups of five. Eight different moisturizers were tested in an environment with a room temperature of (23±2)℃ and a humidity of 40%-60%. One group served as a blank control group.

[0175] Before the experiment, volunteers need to be quiet in the environment for at least 30 minutes. After the environment reaches equilibrium, draw a 4cm×4cm experimental area on each of the left and right forearms, with a minimum interval of 1cm between the areas. The left arm of the subject is the blank control area, and the right arm is the area to be tested.

[0176] Take the homemade moisturizing cream at a concentration of (2.0±0.1) mg / cm³. 2 The appropriate dosage was applied to the test area. After 30 minutes and 2 hours of cosmetic application, technicians used a skin moisture testing probe to measure the moisture content of the test area. The average of five measurements was taken as the test value. The formula for calculating the rate of change in moisture value is:

[0177] Moisture content change rate = (moisture content after use - moisture content before use) / moisture content before use × 100%. The test results are shown in Table 5 below.

[0178] Table 5. Changes in skin moisture levels after 30 minutes and 2 hours following application of homemade moisturizing cream.

[0179]

[0180] As can be seen from the data in the table above, compared with the blank control, the self-made moisturizing cream of this invention has a certain moisturizing effect on the skin.

[0181] After 30 minutes, the skin moisture value changed by more than 20%. The moisturizing agents contained in the moisturizing cream were sodium hyaluronate group, small molecule sodium hyaluronate group, sodium hyaluronate oligosaccharide group, sodium hyaluronate oligosaccharide control group 1, sodium hyaluronate oligosaccharide control group 2, 3.00% aloe vera gel extract, and ceramide group.

[0182] The moisturizing creams that showed a skin moisture change rate of over 20% after 2 hours contained only the following moisturizing agents: small molecule sodium hyaluronate group, sodium hyaluronate oligosaccharide, sodium hyaluronate oligosaccharide control group 1, sodium hyaluronate oligosaccharide control group 2, and ceramide group.

[0183] Moisturizers that showed a skin moisture change rate of over 20% at both 30 minutes and 2 hours included the small molecule sodium hyaluronate group, the sodium hyaluronate oligosaccharide group, the sodium hyaluronate oligosaccharide control group 1, the sodium hyaluronate oligosaccharide control group 2, and the ceramide group.

[0184] Using 4.0% glycerin as the standard, the formula for calculating the moisturizing ability of a humectant is:

[0185] Moisturizing ability = Change rate of moisture value after using moisturizer / Change rate of moisture value after using glycerin. See the appendix for a comparison of the moisturizing abilities of different moisturizers. Figure 4 .

[0186] The experimental results show that different moisturizers have different moisturizing abilities, mainly due to the different moisturizing mechanisms of moisturizers with different molecular weights. Sodium hyaluronate, aloe vera gel extract, and tremella fuciformis extract have larger molecular weights, and their polysaccharide components can form a film on the skin surface, preventing the evaporation of skin moisture in a short time. Small-molecule sodium hyaluronate, sodium hyaluronate oligosaccharide group, sodium hyaluronate oligosaccharide control group 1, sodium hyaluronate oligosaccharide control group 2, and ceramide have smaller molecular weights, which can penetrate deep into the inner layer of the skin and combine with free water in the skin, making it less volatile, and thus have a stronger ability to lock in water and prevent moisture evaporation. Moreover, after entering the skin, the sodium hyaluronate oligosaccharide group, due to the high content of sodium hyaluronate oligosaccharide hexasaccharides to sodium hyaluronate oligosaccharide decasaccharides and relatively large molecular weight, has a slower metabolism in the skin and a stronger water retention capacity. Therefore, sodium hyaluronate oligosaccharides have the best moisturizing effect.

[0187] In summary, the sodium hyaluronate oligosaccharide composition prepared by this invention has the advantages of good moisturizing properties and good transdermal absorption, and is safe to use with no side effects. It can be widely used in food, daily chemical products, medical products and other fields.

[0188] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing a sodium hyaluronate oligosaccharide composition, characterized in that, Includes the following steps: (1) Prepare an acidic aqueous solution using sodium hyaluronate as a raw material and carry out the reaction; The molecular weight of the high molecular weight sodium hyaluronate is 50,000-2,000,000 Da; The acid used to prepare the acidic aqueous solution is sulfuric acid or hydrochloric acid; the concentration of the acid is 0.5-4.0M. The mass ratio of the macromolecular sodium hyaluronate to the acid is 1:5-15; The reaction step is carried out at a temperature of 50-150℃ for 3-15 hours. (2) Adjust the pH of the above reaction solution to neutral, add ethanol and make the ethanol concentration reach 50-70%, and collect the first precipitate and the supernatant separately for later use; (3) Remove impurities from the supernatant by ultrafiltration, add ethanol to make the ethanol concentration reach 70-90%, and collect the precipitated second precipitate; The ultrafiltration membrane has a molecular weight cutoff of 1500-3000 da; (4) Dissolve the second precipitate in water and purify it using a reversed-phase ODS column to obtain the final product.

2. The method for preparing the sodium hyaluronate oligosaccharide composition according to claim 1, characterized in that, In step (4), the purification step includes elution with 0.5-3 v / v% ethanol aqueous solution and elution with 40-60 v / v% ethanol aqueous solution. The elution fraction of 40-60 v / v% ethanol is collected, the ethanol is recovered and dried to obtain the final product.

3. The method for preparing the sodium hyaluronate oligosaccharide composition according to claim 1 or 2, characterized in that, The method further includes the steps of dissolving the first precipitate in water and purifying it with an activated carbon column, collecting the purified product and using the purified product as a raw material to repeat steps (1)-(2). The purification step using an activated carbon column includes elution with water and elution with a 20-40 v / v% ethanol aqueous solution. The elution fraction of the 20-40 v / v% ethanol aqueous solution is collected, the ethanol is recovered, and the product is dried to obtain the desired purified product.

4. The method for preparing the sodium hyaluronate oligosaccharide composition according to claim 1 or 2, characterized in that: The second precipitate was dissolved in water to a concentration of 10-25 mg / mL. The first precipitate was dissolved in water to a concentration of 20-35 mg / mL.

5. A sodium hyaluronate oligosaccharide composition prepared by the method according to any one of claims 1-4, characterized in that, The oligosaccharide composition includes: sodium hyaluronate disaccharide, sodium hyaluronate tetrasaccharide, sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, sodium hyaluronate decasaccharide and sodium hyaluronate dodecasaccharide; Wherein, taking the total amount of the oligosaccharide composition as 100%, The total mass content of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, and sodium hyaluronate decasaccharide is 40-60 wt%. The content of sodium hyaluronate disaccharide is 10-24 wt%, the content of sodium hyaluronate tetrasaccharide is 20-33 wt%, and the content of sodium hyaluronate dodecanose is 3-8 wt%. The mass ratio of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, and sodium hyaluronate decasaccharide is 20-26:13-19:7-17.

6. The sodium hyaluronate oligosaccharide composition according to claim 5, characterized in that, The average molecular weight of the sodium hyaluronate oligosaccharide composition is 1100-1600 Da, and the pH value of a 1 wt% aqueous solution is 6-7.

7. The use of the sodium hyaluronate oligosaccharide composition according to any one of claims 5 or 6, or the sodium hyaluronate oligosaccharide composition prepared by any one of claims 1-5, in the preparation of food, medical products and / or daily chemical products.

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