Calcium hyaluronate, its preparation method and use

Abstract

The application provides a preparation method of calcium hyaluronate, which comprises the following steps: adding sodium hyaluronate into an acidic displacement solution containing calcium salt, performing displacement to obtain calcium hyaluronate precipitate; washing the precipitate with an acidic washing solution; dehydrating the washed precipitate with a neutral dehydration solution, and vacuum drying to obtain calcium hyaluronate powder; wherein the organic medium concentration in the displacement solution is 50-90 wt%, preferably 60-80 wt%, the calcium salt concentration is 0.5-4 wt%, preferably 1-3 wt%, the organic medium concentration in the washing solution is 50-90 wt%, preferably 60-80 wt%, and the organic medium concentration in the dehydration solution is greater than or equal to 90 wt%. The application obtains calcium hyaluronate finished product through high-efficiency displacement, and solves the problems of multiple displacement times, long time consumption, large calcium salt consumption, low calcium displacement efficiency and turbidity of calcium hyaluronate solution in the current calcium hyaluronate production process.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to calcium hyaluronic acid, its preparation method and application. Background Technology

[0002] Calcium hyaluronic acid is the calcium salt of hyaluronic acid, a high-molecular-weight mucopolysaccharide composed of alternating N-acetylglucosamine and D-glucuronic acid disaccharide units. It is widely found in various tissues of the human body and is an important component of skin, vitreous humor, synovial fluid, and soft tissues, possessing unique physicochemical properties and biological functions. Its unique viscoelasticity, water retention, permeability, biocompatibility, and non-immunogenicity make it widely used in ophthalmology, orthopedics, cosmetics, and health foods. In addition to the moisturizing and viscoelastic properties of hyaluronic acid, calcium hyaluronic acid also has good water solubility, making it easily absorbed by the human body. It can be used as a health product or nutritional supplement to supplement calcium, playing a certain role in the prevention of arthritis, enhancing immunity, and preventing osteoporosis. It also has the functions of repairing tissue damage and maintaining joint lubrication, and can be widely used in the food, health product, pharmaceutical, and cosmetic skincare industries.

[0003] There are two existing preparation processes for calcium hyaluronic acid: Patent CN 1563108A describes a method for preparing low molecular weight calcium hyaluronic acid by reducing sodium hyaluronate to a suitable molecular weight, dissolving it, adsorbing it with activated carbon, filtering it, loading the liquid onto a column, performing ion exchange, concentrating it by heating it with a calcium salt solution in a water bath, and finally obtaining the finished calcium hyaluronic acid powder after precipitation, dehydration, and drying. This method is relatively complex, has a low yield, a long cycle, and requires multiple pieces of equipment, making it unsuitable for large-scale workshop production. Patent CN1326883C describes a method for preparing calcium hyaluronic acid by reacting sodium hyaluronate with an organic medium containing calcium salts through a displacement reaction, replacing sodium ions in sodium hyaluronate with calcium ions to produce the finished calcium hyaluronic acid product. This method involves multiple displacement reactions, resulting in low displacement efficiency, the use of a large amount of organic medium, a long cycle, and the potential for residual free calcium ions in the finished product. Furthermore, the weak binding between hyaluronic acid and calcium ions leads to unstable product quality and turbidity in the solution. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides calcium hyaluronic acid, its preparation method, and its applications.

[0005] Specifically, the present invention relates to the following aspects:

[0006] 1. A method for preparing calcium hyaluronic acid, characterized by comprising the following steps:

[0007] Sodium hyaluronate is added to an acidic replacement solution containing calcium salts to perform replacement, resulting in calcium hyaluronate precipitate.

[0008] The precipitate was washed with an acidic detergent.

[0009] The washed precipitate was dehydrated with a neutral dehydrating solution and vacuum dried to obtain calcium hyaluronic acid powder.

[0010] The organic medium concentration in the replacement solution is 50% to 90 wt%, preferably 60% to 80 wt%, and the calcium salt concentration is 0.5% to 4 wt%, preferably 1% to 3 wt%.

[0011] 2. The preparation method according to item 1, characterized in that the organic medium is an organic medium that is compatible with water but insoluble or slightly soluble in sodium hyaluronate or calcium hyaluronate, preferably an alcoholic organic solvent or a ketone organic solvent, more preferably ethanol, methanol or acetone.

[0012] 3. The preparation method according to item 1, characterized in that the molecular weight of sodium hyaluronate is 3kDa to 2200kDa.

[0013] 4. The preparation method according to item 1, characterized in that the mass ratio of sodium hyaluronate to calcium salt is 1:0.5 to 5, preferably 1:1 to 3.

[0014] 5. The preparation method according to item 1, characterized in that the pH of the replacement solution and the washing solution are both 5.0 to 7.0, and the concentration of the organic medium in the washing solution is 50% to 90 wt%, preferably 60% to 80 wt%.

[0015] The concentration of organic media in the dehydration liquid is greater than or equal to 90 wt%.

[0016] 6. The preparation method according to item 1, characterized in that the number of replacements is 1 to 4 times, preferably 1 to 2 times, and the replacement time is 1 to 24 hours, preferably 5 to 16 hours; preferably, the number of washings is 1 to 5 times, preferably 2 to 3 times; more preferably, the number of dehydration times is 1 to 5 times, preferably 2 to 3 times.

[0017] 7. The preparation method according to item 1, wherein the calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium acetate, calcium formate, and calcium nitrite.

[0018] 8. The preparation method according to item 1, characterized in that the calcium hyaluronic acid powder obtained has a calcium hyaluronic acid content of greater than or equal to 90 wt% and a light transmittance of greater than or equal to 99% in a 1 wt% aqueous solution.

[0019] 9. A calcium hyaluronic acid, prepared by any one of items 1-8.

[0020] 10. The calcium hyaluronic acid according to item 9, characterized in that the calcium hyaluronic acid is used for damage repair, increasing the water content of the stratum corneum of the skin, and reducing fine lines on the skin.

[0021] This invention obtains calcium hyaluronic acid through highly efficient replacement, solving the problems of numerous replacement cycles, long processing time, high calcium salt consumption, low calcium replacement efficiency, and turbidity of the calcium hyaluronic acid solution in current calcium hyaluronic acid production processes. Specifically, the yield of calcium hyaluronic acid prepared by this invention is above 93.75%, and the light transmittance is above 98.5%. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present invention, and are not intended to limit the present invention.

[0023] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The invention is further described below with reference to specific embodiments, but is not intended to limit the scope of the invention.

[0024] This invention provides a method for preparing calcium hyaluronic acid, characterized by comprising the following steps:

[0025] Sodium hyaluronate is added to an acidic replacement solution containing calcium salts to perform replacement, resulting in calcium hyaluronate precipitate.

[0026] The precipitate was washed with an acidic detergent.

[0027] The washed precipitate was dehydrated with a neutral dehydrating solution and vacuum dried to obtain calcium hyaluronic acid powder.

[0028] The acidic replacement solution containing calcium salts is obtained by filtration through an organic filter membrane, wherein the pore size of the filter membrane is required to be 0.2 to 3 micrometers, preferably 0.22 to 1.2 micrometers.

[0029] The replacement solution is acidic and comprises calcium salts, an organic medium, and water. The pH of the replacement solution can be adjusted using an acid that does not affect the replacement, such as glacial acetic acid. The concentration of the organic medium in the replacement solution is 50%–90 wt%, for example, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, or 90 wt%, preferably 60%–80 wt%. The concentration of the calcium salt is 0.5%–4 wt%, for example, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, or 4 wt%, preferably 1%–3 wt%.

[0030] The organic medium is an organic medium that is compatible with water but insoluble or only slightly soluble in sodium hyaluronate or calcium hyaluronate. It is preferably an alcoholic organic solvent or a ketone organic solvent, and more preferably ethanol, methanol or acetone.

[0031] The calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium acetate, calcium formate, and calcium nitrite.

[0032] The molecular weight of sodium hyaluronate in the replacement step can be adjusted according to the molecular weight of the calcium hyaluronate to be prepared. In one specific embodiment, the molecular weight of the sodium hyaluronate is 3kDa to 2200kDa, for example, it can be 3kDa, 10kDa, 50kDa, 100kDa, 200kDa, 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, etc.

[0033] In one specific embodiment, the organic medium is ethanol.

[0034] In one specific embodiment, the pH of the replacement solution is 5.0 to 7.0, for example, it can be 5.0, 5.5, 6.0, 6.5, 6.8, 6.9, etc.

[0035] Displacement is typically performed under stirring. The number of displacements and the duration can be adjusted as needed. In one specific embodiment, the number of displacements is 1 to 4, for example, 1, 2, 3, or 4 times, preferably 1 to 2 times. The displacement time is 1 to 24 hours, for example, 1 hour, 2 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 24 hours, preferably 5 to 16 hours.

[0036] The washing solution is acidic and includes organic media and water. The pH of the washing solution can be adjusted using an acid that does not affect washing, such as glacial acetic acid. The concentration of organic media in the washing solution is 50% to 90 wt%, for example, 50 wt%, 55 wt%, 60 wt%, 65 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, or 90 wt%, preferably 60% to 80 wt%.

[0037] As described above, in one specific embodiment, the organic medium is ethanol.

[0038] In one specific embodiment, the pH of the washing solution is 5.0 to 7.0, for example, it can be 5.0, 5.5, 6.0, 6.5, 6.8, 6.9, etc.

[0039] The volume ratio of the washing solution to the calcium hyaluronic acid precipitate is 1 to 3:1, for example, 1:1, 2:1, or 3:1, preferably 1 to 2:1.

[0040] The number of washes can be adjusted as needed. In one specific embodiment, the number of washes is 1 to 5 times, for example, 1, 2, 3, 4, or 5 times, preferably 2 to 3 times.

[0041] The dehydration solution is neutral and comprises an organic medium and water. The concentration of the organic medium in the dehydration solution is greater than or equal to 90 wt%, for example, it can be 90 wt%, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, 97 wt%, 98 wt%, or 99 wt%.

[0042] The number of dehydration cycles can be adjusted as needed. In one specific embodiment, the number of dehydration cycles is 1 to 5, for example, 1, 2, 3, 4, or 5 times, preferably 2 to 3 times.

[0043] In one specific embodiment, the molecular weight of the calcium hyaluronic acid obtained by the above method is 2kDa to 2000kDa.

[0044] In one specific embodiment, the calcium ion content in the obtained calcium hyaluronic acid powder is 3.0 to 10.0 wt%, for example, it can be 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, or 10 wt%.

[0045] In one specific embodiment, the obtained calcium hyaluronic acid powder has a transmittance of greater than or equal to 99% in a 1 wt% aqueous solution, for example, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, etc.

[0046] In one specific embodiment, the calcium hyaluronic acid powder obtained contains calcium hyaluronic acid content greater than or equal to 90 wt%, for example, 90 wt%, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, 97 wt%, 98 wt%, 99 wt%, etc.

[0047] In one specific embodiment, the sodium ion content in the obtained calcium hyaluronic acid powder is less than or equal to 3%, for example, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.8%, 2.0%, 2.8%, 3.0%, etc.

[0048] In this invention, the molecular weight of calcium hyaluronic acid is determined by the intrinsic viscosity method. The specific determination method is as follows.

[0049] Preparation of test solution: Accurately weigh approximately 100 mg (W1) of the test sample (Note: This sample amount is uncertain; the sample amount should be increased when the intrinsic viscosity is less than 3.0 dL / g or the molecular weight is less than 100,000) into a 100 ml volumetric flask, add 0.2 mol / L sodium chloride solution to make up to volume, and shake well; then weigh a certain amount of this solution (W2) (Note: This sample amount is uncertain and should be adjusted according to the eluent time of the test solution) into a 100 ml volumetric flask, add 0.2 mol / L sodium chloride solution to make up to volume, and shake well to obtain the test solution.

[0050] Determination of solvent outflow time (t0): Take an appropriate amount of filtered 0.2 mol / L sodium chloride solution, discard the initial filtrate, and inject the subsequent filtrate (not less than 7 ml) into a clean and dry Ubbelohde viscometer. Fix the viscometer vertically in a constant temperature water bath (temperature 25℃ ± 0.1℃) for 15 minutes, accurately record the outflow time, repeat the measurement at least twice, and take the average value as the outflow time (t0) of the blank solution.

[0051] Determination of test solution outflow time (t1): Take an appropriate amount of test solution and measure it according to the method for determining solvent outflow time. Repeat the measurement at least twice. The difference between the two measurements should not exceed 0.2 seconds. Take the average value as the outflow time (t1) of the test solution.

[0052] The ratio t1 / t0 should be between 1.3 and 1.5; otherwise, the concentration of the test solution should be adjusted before testing.

[0053] The concentration C (g / dL) of the test solution is calculated using the following formula:

[0054]

[0055] ρ 25 = 1.000 g / ml (density of the test solution at 25°C)

[0056] The intrinsic viscosity is calculated using the following formula:

[0057]

[0058] Calculate the average molecular weight of calcium hyaluronic acid:

[0059] Intrinsic viscosity [η] (dL / g) = 3.6 × 10⁻⁶ -4 M 0.78 (M: Average molecular weight)

[0060]

[0061] In this invention, the transmittance of the obtained calcium hyaluronic acid is determined by light absorption method. The specific determination method is as follows.

[0062] Take 0.1g of sample into an Erlenmeyer flask containing 100ml of water, place it in the refrigerator overnight to dissolve, pour the sample solution into a 1ml cuvette, use water as a blank, and measure the transmittance of the solution at a wavelength of 550nm.

[0063] In this invention, the calcium content of the obtained calcium hyaluronic acid is determined by flame atomic absorption spectrometry. The specific determination method is as follows.

[0064] Preparation of standard curve:

[0065] Accurately weigh 1 ml of calcium single-element solution standard (1000 μg / ml) into a 100 ml volumetric flask, and dilute to volume with 1% nitric acid to obtain a standard stock solution (10 μg / ml). Measure 0, 0.2, 0.4, 0.8, and 1.6 ml of the standard stock solution into five clean 10 ml volumetric flasks, and dilute to volume with 1% nitric acid. This yields calcium standard solutions with calcium concentrations of 0, 0.2, 0.4, 0.8, and 1.6 μg / ml, respectively.

[0066] Test solution: Accurately weigh 50 mg of the test sample into a 100 ml volumetric flask, add 1 ml of nitric acid, heat in a water bath until completely dissolved, dilute to volume with water, take 1 ml of the above solution into a 50 ml volumetric flask, and dilute to volume with 1% nitric acid to obtain the test solution.

[0067] Determination: After zeroing with a blank solution, take appropriate amounts of each solution and measure the absorbance using flame atomic absorption spectrometry at a detection wavelength of 213.9 nm. Perform linear regression of the absorbance (Y) of the test solution against the calcium concentration (X, μg / ml) to plot a standard curve. Calculate the calcium concentration in the test solution using the following formula.

[0068]

[0069] In the formula, C i —Calcium concentration in the test solution, μg / ml;

[0070] W—Sample weight of the test sample, mg;

[0071] h% — Loss on drying of the test sample.

[0072] In this invention, the uronic acid content is detected using the sulfuric acid-carbazole colorimetric method.

[0073] In this invention, the yield is the ratio of the output to the input, and the calculation formula is as follows:

[0074] Yield = Calcium hyaluronic acid output / Sodium hyaluronic acid input × 100%

[0075] The present invention also provides calcium hyaluronic acid prepared by the above method.

[0076] The calcium hyaluronic acid has all the functions of hyaluronic acid, such as damage repair, increasing the moisture content of the stratum corneum, and reducing fine lines.

[0077] Example 1

[0078] Prepare 2.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution using 100kg of calcium chloride. Adjust the pH of the ethanol to 5.6 using glacial acetic acid, resulting in a replacement solution concentration of 80wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0079] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 1480 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 24 hours.

[0080] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an ethanol concentration of 78% and a pH of 5.6 is added for washing. The washing is performed once, and each washing takes 3 hours.

[0081] After washing, the excess supernatant is extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate is added for dehydration. The dehydration time is 0.5–1 hour, and the process is repeated 3 times. The alcohol content of the supernatant is maintained above 90%. The supernatant is then transferred to a three-in-one dryer for vacuum drying at 45°C, a vacuum degree of 0.10 MPa, and a drying time of 20 hours. The resulting product is 19.4 kg of calcium hyaluronic acid.

[0082] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 7.2%, the sodium content was 0.23%, the yield was 97%, the molecular weight was 1.27 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.5%, and the uronic acid content was 48.16%.

[0083] Example 2

[0084] Prepare 1.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution. Use 60kg of calcium chloride and adjust the pH of the ethanol to 6.2 with glacial acetic acid. The concentration of the replacement solution is 80wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0085] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 1480 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 24 hours.

[0086] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an ethanol concentration of 75% and a pH of 5.8 is added for washing. The washing is performed once, and each washing takes 3 hours.

[0087] After washing, the excess supernatant is extracted, and a dehydrating solution with a concentration of 90% ethanol, equal in volume to the precipitate, is added for dehydration. The dehydration time is 0.5–1 hour, and the process is repeated 3 times. The alcohol content of the supernatant is maintained above 90%. The supernatant is then transferred to a three-in-one dryer for vacuum drying at 45°C and a vacuum degree of 0.10 MPa for 22 hours. The resulting product is 19.2 kg of calcium hyaluronic acid.

[0088] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 6.8%, the sodium content was 0.35%, the yield was 96%, the molecular weight was 1.3 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.3%, and the uronic acid content was 47.93%.

[0089] Example 3

[0090] Prepare 0.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution using 20kg of calcium chloride. Adjust the pH of the ethanol to 6.0 using glacial acetic acid, resulting in a replacement solution concentration of 80wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0091] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 1480 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 24 hours.

[0092] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an ethanol concentration of 78% and a pH of 6.20 is added for washing. The washing is performed once, and each washing takes 3 hours.

[0093] After washing, the excess supernatant was extracted, and a dehydration solution with a concentration of 90% ethanol, equal in volume to the precipitate, was added for dehydration. The dehydration time was 0.5–1 hour, and the process was repeated 3 times. The alcohol content of the supernatant was maintained above 90%. The supernatant was then transferred to a three-in-one dryer for vacuum drying at 45°C and a vacuum degree of 0.10 MPa for 22 hours. The resulting product was 19.27 kg of calcium hyaluronic acid.

[0094] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 5.0%, the sodium content was 0.54%, the yield was 96.35%, the molecular weight was 1.32 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.4%, and the uronic acid content was 48.54%.

[0095] Example 4

[0096] Prepare 0.25T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution with 20Kg of calcium chloride. Adjust the pH of the ethanol to 5.8 using glacial acetic acid, resulting in a replacement solution concentration of 75wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0097] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 1360 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 24 hours.

[0098] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 72% and a pH of 6.2. The washing is performed once, and each washing takes 3 hours.

[0099] After washing, the excess supernatant is extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate is added for dehydration. The dehydration time is 0.5–1 hour, and the process is repeated 3 times. The alcohol content of the supernatant is maintained above 90%. The supernatant is then transferred to a three-in-one dryer for vacuum drying at 45°C, a vacuum degree of 0.10 MPa, and a drying time of 23 hours. The resulting product is 19.30 kg of calcium hyaluronic acid.

[0100] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 4.8%, the sodium content was 0.62%, the yield was 96.51%, the molecular weight was 1.15 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.2%, and the uronic acid content was 48.32%.

[0101] Example 5

[0102] Prepare 1.8T of 3.0wt% calcium chloride-ethanol (acidic) replacement solution, using 60Kg of calcium chloride. Adjust the pH of the ethanol to 6.5 using glacial acetic acid, resulting in a replacement solution concentration of 80wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0103] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 320 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 24 hours.

[0104] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of washing solution is added for washing. The washing solution has an ethanol concentration of 75% and a pH of 6.32. The washing is performed once, and each washing time is 3 hours.

[0105] After washing, the excess supernatant is extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate is added for dehydration. The dehydration time is 0.5–1 hour, and the dehydration is repeated 3 times. The alcohol content of the supernatant is ensured to be above 90%. Then, it is transferred to a three-in-one dryer for vacuum drying at a temperature of 45℃, a vacuum degree of 0.10 MPa, and a drying time of 20 hours. 19.34 kg of calcium hyaluronic acid is obtained from the output.

[0106] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 5.6%, the sodium content was 0.32%, the yield was 96.7%, the molecular weight was 290,000, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.7%, and the uronic acid content was 48.33%.

[0107] Example 6

[0108] Prepare 1.8T of 3.0wt% calcium acetate-ethanol (acidic) replacement solution, using 60Kg of calcium acetate. Adjust the pH of the ethanol to 6.5 using glacial acetic acid, resulting in a replacement solution concentration of 80wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0109] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 320 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 16 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 16 hours.

[0110] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 78% and a pH of 5.82. The washing is performed once, and each washing takes 3 hours.

[0111] After washing, the excess supernatant is extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate is added for dehydration. The dehydration time is 0.5–1 hour, and the process is repeated 3 times. The alcohol content of the supernatant is maintained above 90%. The supernatant is then transferred to a three-in-one dryer for vacuum drying at 45°C, a vacuum degree of 0.10 MPa, and a drying time of 22 hours. The resulting product is 19.28 kg of calcium hyaluronic acid.

[0112] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 5.5%, the sodium content was 0.35%, the yield was 96.4%, the molecular weight was 270,000, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.5%, and the uronic acid content was 48.41%.

[0113] Example 7

[0114] Prepare 2.0T of 4.0wt% calcium nitrate-ethanol (acidic) replacement solution, using 60Kg of calcium nitrate. Adjust the pH of the ethanol to 6.8 using glacial acetic acid, resulting in a replacement solution concentration of 70wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0115] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 320 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 24 hours.

[0116] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 78% and a pH of 5.82. The washing is performed once, and each washing takes 3 hours.

[0117] After washing, the excess supernatant is extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate is added for dehydration. The dehydration time is 0.5–1 hour, and the process is repeated 3 times. The alcohol content of the supernatant is maintained above 90%. The supernatant is then transferred to a three-in-one dryer for vacuum drying at 45°C, a vacuum degree of 0.10 MPa, and a drying time of 22 hours. The resulting product is 19.28 kg of calcium hyaluronic acid.

[0118] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 6.2%, the sodium content was 0.34%, the yield was 96.4%, the molecular weight was 250,000, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.5%, and the uronic acid content was 48.76%.

[0119] Example 8

[0120] Prepare 5 tons of 2.0 wt% calcium chloride-ethanol (acidic) replacement solution using 100 kg of calcium chloride, adjust the pH of the ethanol to 5.5 with hydrochloric acid, and the concentration of the replacement solution is 76 wt%. Filter the solution through a 0.45 micron polypropylene membrane for later use.

[0121] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 108 kDa) and add it to the replacement solution. Start stirring to perform replacement for 5 hours. Stop stirring and let stand for about 1-2 hours. Remove the excess supernatant, add the remaining replacement solution, and continue the second replacement. Perform a total of 2 replacements, each lasting about 5 hours.

[0122] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 77% and a pH of 5.93. The washing is performed once, and each washing takes 3 hours.

[0123] After washing, the excess supernatant was extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate was added for dehydration. The dehydration time was 0.5–1 hour, and the process was repeated 3 times. The alcohol content of the supernatant was ensured to be above 90%. Then, the supernatant was transferred to a three-in-one dryer for vacuum drying at a temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 21 hours. 18.84 kg of calcium hyaluronic acid was obtained from the output.

[0124] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 6.7%, the sodium content was 0.39%, the yield was 94.18%, the molecular weight was 990,000, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.4%, and the uronic acid content was 48.16%.

[0125] Example 9

[0126] Prepare 2T of 1.0wt% calcium chloride-ethanol (acidic) replacement solution, using 20Kg of calcium chloride. Adjust the pH of the ethanol to 6.5 using glacial acetic acid, resulting in a replacement solution concentration of 75wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0127] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 110 kDa) and add it to the replacement solution. Start stirring to perform replacement for 5 hours. Stop stirring and let stand for about 1-2 hours. Remove the excess supernatant, add the remaining replacement solution, and continue the second replacement. Perform a total of 2 replacements, each lasting about 5 hours.

[0128] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 72% and a pH of 5.99. The washing is performed twice, and each washing time is 3 hours.

[0129] After washing, the excess supernatant is extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate is added for dehydration. The dehydration time is 0.5–1 hour, and the dehydration is repeated 3 times. The alcohol content of the supernatant is ensured to be above 90%. Then, it is transferred to a three-in-one dryer for vacuum drying at a temperature of 45℃, a vacuum degree of 0.10 MPa, and a drying time of 22 hours. 18.35 kg of calcium hyaluronic acid is obtained from the output.

[0130] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 5.3%, the sodium content was 0.65%, the yield was 93.75%, the molecular weight was 1.02 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.5%, and the uronic acid content was 48.18%.

[0131] Example 10

[0132] Prepare 10T of 1wt% calcium acetate-ethanol (acidic) replacement solution, using 10Kg of calcium acetate. Adjust the pH of the ethanol to 5.2 using glacial acetic acid, resulting in a replacement solution concentration of 80wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0133] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 30 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 1 hour. Stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process twice, each time for about 3 hours.

[0134] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of washing solution is added for washing. The washing solution has an ethanol concentration of 79% and a pH of 6.07. The washing is performed twice, and each washing time is 3 hours.

[0135] After washing, the excess supernatant was extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate was added for dehydration. The dehydration time was 0.5–1 hour, and the process was repeated 3 times. The alcohol content of the supernatant was ensured to be above 90%. Then, the supernatant was transferred to a three-in-one dryer for vacuum drying at a temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 21 hours. 19.78 kg of calcium hyaluronic acid was obtained from the output.

[0136] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 3.6%, the sodium content was 1.8%, the yield was 96.1%, the molecular weight was 47,000, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.4%, and the uronic acid content was 48.79%.

[0137] Example 11

[0138] Prepare 10T of 1wt% calcium chloride-ethanol (acidic) replacement solution, using 10Kg of calcium chloride. Adjust the pH of the ethanol to 5.8 using glacial acetic acid, resulting in a replacement solution concentration of 78wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0139] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 450 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 1 hour. Stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 1 hour.

[0140] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 78% and a pH of 6.21. The washing is performed once, and each washing takes 3 hours.

[0141] After washing, the excess supernatant was extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate was added for dehydration. The dehydration time was 0.5–1 hour, and the process was repeated 3 times. The alcohol content of the supernatant was ensured to be above 90%. Then, the supernatant was transferred to a three-in-one dryer for vacuum drying at a temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 21 hours. 19.67 kg of calcium hyaluronic acid was obtained from the output.

[0142] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 3.5%, the sodium content was 1.4%, the yield was 98.35%, the molecular weight was 39,000, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.4%, and the uronic acid content was 48.34%.

[0143] Example 12

[0144] Prepare 1.5T of 4wt% calcium chloride-ethanol (acidic) replacement solution with 60Kg of calcium chloride. Adjust the pH of the ethanol to 6.3 using glacial acetic acid, resulting in a replacement solution concentration of 90wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0145] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 8.6 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 20 hours. Stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 20 hours.

[0146] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 78% and a pH of 6.21. The washing is performed twice, and each washing time is 3 hours.

[0147] After washing, the excess supernatant was extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate was added for dehydration. The dehydration time was 0.5–1 hour, and the process was repeated 3 times. The alcohol content of the supernatant was ensured to be above 90%. Then, the supernatant was transferred to a three-in-one dryer for vacuum drying at a temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 21 hours. 19.47 kg of calcium hyaluronic acid was obtained from the output.

[0148] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 6.5%, the sodium content was 0.19%, the yield was 97.36%, the molecular weight was 0.98 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 98.5%, and the uronic acid content was 48.14%.

[0149] Comparative Example 1

[0150] Prepare 1 ton of 6 wt% calcium chloride-ethanol (acidic) replacement solution using 60 kg of calcium chloride. Adjust the pH of the ethanol to 5.9 using glacial acetic acid, resulting in a replacement solution concentration of 55 wt%. Filter the solution through a 0.45 micron polypropylene membrane for later use.

[0151] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 1570 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 0.5 h. Stop stirring and let it stand for about 1-2 h. Remove the excess supernatant. Repeat the replacement process once, each time for about 0.5 h.

[0152] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 50% and a pH of 4.3. The washing is performed once, and each washing takes 3 hours.

[0153] After washing, the excess supernatant is extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate is added for dehydration. The dehydration time is 0.5–1 hour, and the process is repeated 3 times. The alcohol content of the supernatant is maintained above 90%. The supernatant is then transferred to a three-in-one dryer for vacuum drying at 45°C, a vacuum degree of 0.10 MPa, and a drying time of 20 hours. The resulting product is 19.02 kg of calcium hyaluronic acid.

[0154] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 3.2%, the sodium content was 2.8%, the yield was 95.1%, the molecular weight was 1.38 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 85.7%, and the uronic acid content was 46.56%.

[0155] Comparative Example 2

[0156] Prepare 1.5T of 4wt% calcium chloride-ethanol (acidic) replacement solution with 6Kg of calcium chloride. Adjust the pH of the ethanol to 5.6 using glacial acetic acid, resulting in a replacement solution concentration of 70wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0157] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 1650 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. Stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process twice, each time for about 24 hours.

[0158] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 75% and a pH of 6.45. The washing is performed once, and each washing takes 3 hours.

[0159] After washing, the excess supernatant was extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate was added for dehydration. The dehydration time was 0.5–1 hour, and the process was repeated 3 times. The alcohol content of the supernatant was ensured to be above 90%. Then, the supernatant was transferred to a three-in-one dryer for vacuum drying at a temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 23 hours. 19.13 kg of calcium hyaluronic acid was obtained from the output.

[0160] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 2.72%, the sodium content was 3.2%, the yield was 95.64%, the molecular weight was 1.15 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.1%, and the uronic acid content was 47.17%.

[0161] Comparative Example 3

[0162] Prepare 1.8T of 3wt% calcium chloride-ethanol (acidic) replacement solution with 60Kg of calcium chloride. Adjust the pH of the ethanol to 7.5 using glacial acetic acid, resulting in a replacement solution concentration of 76wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0163] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 380 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 5 hours. Stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process twice, each time for about 5 hours.

[0164] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an equal volume of precipitate is added for washing. The washing solution has an ethanol concentration of 75% and a pH of 6.45. The washing is performed once, and each washing takes 3 hours.

[0165] After washing, the excess supernatant was extracted, and a 90% ethanol dehydration solution with a volume equal to the volume of the precipitate was added for dehydration. The dehydration time was 0.5–1 hour, and the process was repeated 3 times. The alcohol content of the supernatant was ensured to be above 90%. Then, the supernatant was transferred to a three-in-one dryer for vacuum drying at a temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 23 hours. 19.46 kg of calcium hyaluronic acid was obtained from the output.

[0166] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 6.9%, the sodium content was 0.62%, the yield was 97.3%, the molecular weight was 220,000, the transmittance of the 0.1% calcium hyaluronic acid solution was 88.1%, and the uronic acid content was 48.67%.

[0167] Comparative Example 4

[0168] Prepare 1.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution. Use 60kg of calcium chloride and adjust the pH of the ethanol to 6.2 with glacial acetic acid. The concentration of the replacement solution is 45wt%. Filter the solution through a 0.45-micron polypropylene membrane for later use.

[0169] Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 1480 kDa) and add it to the replacement solution. Start stirring to carry out the replacement for 24 hours. After one replacement is completed, stop stirring and let it stand for about 1-2 hours. Remove the excess supernatant. Repeat the replacement process once, each time for about 24 hours.

[0170] After the replacement is completed, the supernatant is extracted after standing, and a washing solution with an ethanol concentration of 75% and a pH of 5.8 is added for washing. The washing is performed once, and each washing takes 3 hours.

[0171] After washing, the excess supernatant is extracted, and a dehydration solution with a concentration of 90% ethanol, equal in volume to the volume of the precipitate, is added for dehydration. The dehydration time is 0.5–1 hour, and the process is repeated 3 times. The alcohol content of the supernatant is maintained above 90%. The supernatant is then transferred to a three-in-one dryer for vacuum drying at 45°C, a vacuum degree of 0.10 MPa, and a drying time of 22 hours. The resulting product is 16.7 kg of calcium hyaluronic acid.

[0172] The test results showed that the calcium ion content of the sample calcium hyaluronic acid was 6.5%, the sodium content was 0.81%, the yield was 83.5%, the molecular weight was 1.3 million, the transmittance of the 0.1% calcium hyaluronic acid solution was 99.2%, and the uronic acid content was 47.75%.

[0173] The reaction conditions of the above embodiments and comparative examples are shown in Table 1, and the detection results of calcium hyaluronic acid obtained in each embodiment and comparative example are shown in Table 2.

[0174] Table 1. Conditions in the preparation methods of the examples and comparative examples.

[0175]

[0176]

[0177] Table 2. Test results of calcium hyaluronic acid obtained from the examples and comparative examples.

[0178]

[0179]

[0180] The results above show that, as seen in Examples 2 and 5 and Comparative Examples 1 and 4, if the calcium salt concentration is too high, the ethanol concentration needs to be reduced to improve the calcium salt solubility. If the ethanol concentration is too low, the yield of calcium hyaluronic acid will be low. As seen in Examples 4 and Comparative Example 2, even if the calcium salt concentration remains unchanged and the alcohol content of the replacement solution is similar, if the calcium salt concentration is too low in the ratio of sodium hyaluronate to calcium salt, even if the number of replacements is increased, the calcium content in the product will still be low and the sodium content will be high. As seen in Examples 6 and Comparative Example 3, if the pH of the replacement solution is too high, the solubility of calcium salt will decrease, and the final product solution will be turbid.

[0181] Test case

[0182] Experimental Example 1: Damage Repair Efficacy of Calcium Hyaluronic Acid

[0183] (1) Solution preparation. The calcium hyaluronic acid prepared in Example 12 was dissolved in serum-free 1640 culture medium, filtered through a 0.22 μm filter membrane for sterilization, and the concentration was adjusted to 0.025%.

[0184] (2) Simulated scratches. The ibidi chambers are attached to the wells of a 24-well plate near the center, ensuring all chambers are oriented in the same direction. The bottom of the chamber's sidewall is in close contact with the culture plate, preventing cell growth and creating simulated scratches.

[0185] (3) Cell seeding. L929 cell density was adjusted to 1×10⁻⁶. 5 / mL, inoculate 70μL into each of the two wells of the ibidi chamber, add 200μL of serum-containing 1640 culture medium outside the chamber, and incubate at 37℃ and 5% CO2 for 24h.

[0186] (4) Drug administration. Remove the ibidi chamber, discard the culture medium, and add serum-free culture medium containing calcium hyaluronic acid.

[0187] (5) Data processing. Continue culturing for 24 hours and calculate the repair area using ImageJ software.

[0188] Cell migration rate (%) = (1 - area of ​​scratched region after drug application / area of ​​initial scratched region) × 100

[0189] Results and analysis:

[0190] As shown in Table 3, compared with the model group, calcium hyaluronic acid at a concentration of 0.025% exhibited the effect of promoting cell migration and wound healing. The healing rate was equal to the cell migration rate.

[0191] Table 3. Effects of calcium hyaluronic acid on damage repair

[0192]

[0193] Wherein, T0 scratch area represents the initial scratch area, and T24h scratch area represents the value after 24 hours of using calcium hyaluronic acid.

[0194] Experimental Example 2: Evaluation of the efficacy of calcium hyaluronic acid on human skin

[0195] Ten healthy participants were recruited to participate in the test using a half-face control method.

[0196] The left cheek was treated with an emulsion containing 0.5% calcium hyaluronic acid prepared in Example 12, while the right cheek was treated with a blank emulsion.

[0197] The stratum corneum water content, transepidermal water loss, and texture area of ​​the subject's facial apple cheek area were measured before and after using the calcium hyaluronic acid product.

[0198] Thirty minutes after cleansing, the initial values ​​of stratum corneum moisture content, transepidermal water loss, and texture area in the apple cheek area of ​​the left and right sides of the face were measured. Then, equal amounts of calcium hyaluronic acid emulsion and blank emulsion were used once in the morning and once in the evening, and all indicators were measured again at 1, 2 and 4 weeks after use.

[0199] The stratum corneum moisture content was measured using a Corneometer CM 825 (Courage+Khazaka, Germany).

[0200] Transdermal water loss was measured using a skin water loss meter. The test was conducted by TM Hex (Courage+Khazaka, Germany).

[0201] Texture area was detected using the VISIACR facial image analysis system (Canfield, USA).

[0202] The results are shown in Tables 4-6.

[0203] Table 4. Effect of calcium hyaluronic acid on the water content of the stratum corneum

[0204] T0(％) T1W (%) T2W (%) T4W (%) Calcium hyaluronic acid 100.00 132.55 145.47 133.83 blank 100.00 131.41 135.62 128.60

[0205] Wherein, T0 represents the initial moisture content, expressed as 100%; T1W represents the ratio of the stratum corneum moisture content to the initial moisture content after 1 week of using calcium hyaluronic acid; T2W represents the ratio of the stratum corneum moisture content to the initial moisture content after 2 weeks of using calcium hyaluronic acid; and T4W represents the ratio of the stratum corneum moisture content to the initial moisture content after 4 weeks of using calcium hyaluronic acid.

[0206] The results showed that, compared with the control group, the stratum corneum moisture content was higher (increased by 5%-10%) 1-4 weeks after use of calcium hyaluronic acid, indicating that calcium hyaluronic acid can increase the moisture content of the stratum corneum of the skin.

[0207] Table 5. Effect of calcium hyaluronic acid on transepidermal water loss.

[0208] T0(％) T1W (%) T2W (%) T4W (%) Calcium hyaluronic acid 100.00 81.05 93.27 98.13 blank 100.00 92.37 98.45 98.98

[0209] Wherein, T0 represents the initial moisture content, expressed as 100%; T1W represents the ratio of the stratum corneum moisture content to the initial moisture content after 1 week of using calcium hyaluronic acid; T2W represents the ratio of the stratum corneum moisture content to the initial moisture content after 2 weeks of using calcium hyaluronic acid; and T4W represents the ratio of the stratum corneum moisture content to the initial moisture content after 4 weeks of using calcium hyaluronic acid.

[0210] The results showed that, compared with the control group, the transdermal water loss was significantly reduced within 2 weeks after the use of calcium hyaluronic acid, especially by 11% in the first week. This indicates that calcium hyaluronic acid can reduce transdermal water loss.

[0211] Table 6. Effects of calcium hyaluronic acid on fine lines in the skin.

[0212] T0(％) T1W (%) T2W (%) T4W (%) Calcium hyaluronic acid 100.00 111.52 112.89 112.15 blank 100.00 111.56 114.31 118.66

[0213] Where T0 represents the initial texture area, expressed as 100%, T1W represents the ratio of the texture area after 1 week of using calcium hyaluronic acid to the initial texture area, T2W represents the ratio of the texture area after 2 weeks of using calcium hyaluronic acid to the initial texture area, and T4W represents the ratio of the texture area after 4 weeks of using calcium hyaluronic acid to the initial texture area.

[0214] The results showed that calcium hyaluronic acid began to reduce fine lines after 2 weeks of use, and by 4 weeks, the difference between the two groups was significant (p<0.05), with a 6% reduction in the area of ​​fine lines compared to the control group. This indicates that calcium hyaluronic acid has the effect of reducing fine lines.

Claims

1. A method for preparing calcium hyaluronate, characterized by, The method comprises the following steps: adding sodium hyaluronate into an acidic displacement solution containing calcium salt, and performing displacement to obtain a calcium hyaluronate precipitate; washing the precipitate with an acidic washing solution; dehydration of the washed precipitate with a neutral dehydration solution, and vacuum drying to obtain a calcium hyaluronate powder; the concentration of the organic medium in the displacement solution is 80-90wt%, and the concentration of the calcium salt is 3.5-4wt%; the pH of the displacement solution is greater than or equal to 5 and less than 7; the organic medium is an organic medium which is compatible with water, but in which sodium hyaluronate or calcium hyaluronate is insoluble or slightly soluble; the mass ratio of sodium hyaluronate to calcium salt is 1:1-5; the displacement is performed 1-2 times.

2. The production method according to claim 1, characterized by, the organic medium is an alcohol organic solvent or a ketone organic solvent.

3. The method of claim 2, wherein, the organic medium is ethanol, methanol or acetone.

4. The method of claim 1, wherein, the molecular weight of sodium hyaluronate is 3kDa-2200kDa.

5. The preparation method according to claim 1, characterized in that, the mass ratio of sodium hyaluronate to calcium salt is 1:1-3.

6. The method of claim 1, wherein, the pH of the washing solution is greater than or equal to 5 and less than 7, the concentration of the organic medium in the washing solution is 50-90wt%, and the concentration of the organic medium in the dehydration solution is greater than or equal to 90wt%.

7. The production method according to claim 6, wherein the concentration of the organic medium in the washing solution is 60-80wt%.

8. The method of claim 1, wherein, the displacement time is 5-16h.

9. The method of claim 1, wherein, the washing is performed 1-5 times.

10. The method of claim 9, wherein, the washing is performed 2-3 times.

11. The method of claim 1, wherein, the dehydration is performed 1-5 times.

12. The method of claim 11, wherein, the dehydration is performed 2-3 times.

13. The method of claim 1, wherein, the calcium salt is selected from one or more than two of calcium chloride, calcium nitrate, calcium acetate, calcium formate and calcium nitrite.

14. The method of claim 1, wherein, the content of calcium hyaluronate in the obtained calcium hyaluronate powder is greater than or equal to 90wt%, and the light transmittance in a 0.1wt% aqueous solution is greater than or equal to 99%.

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