Preparation method and use of zinc hyaluronate

Abstract

The present application provides a method for preparing zinc hyaluronate, which includes the steps of adding sodium hyaluronate to an acidic aqueous organic medium containing zinc salt, carrying out a single substitution to obtain zinc hyaluronate precipitate; washing the precipitate with a washing solution; dehydrating the washed precipitate with a dehydrating solution and drying to obtain zinc hyaluronate powder. The present application adopts a solid substitution method, and optimizes the concentration of zinc salt in the substitution solution, the volume of the substitution solution, the substitution time and the number of substitutions, thereby solving the problems of the current zinc hyaluronate preparation process having many substitutions, a long time, a large consumption of molecular weight and a low utilization rate of zinc salt, greatly shortening the production cycle, improving the substitution efficiency, shortening the contact time between the raw material and the acidic substitution solution, and avoiding a significant decrease in the molecular weight of the product. The present application provides a method for producing zinc hyaluronate quickly and efficiently, which is suitable for large-scale production of zinc hyaluronate.

Description

[Technical Field]

[0001] The present application relates to the preparation method and use of zinc hyaluronate, which belongs to the technical field of hyaluronate. [Background technology]

[0002] Hyaluronic acid (HA) is a polysaccharide composed of disaccharide units of glucuronic acid and acetylglucosamine, and has physiological functions such as moisturizing, nutrition, and damage repair and prevention, and is widely used in the field of cosmetics. Research has shown that in aqueous solutions of pH 6.0-6.5, Zn 2+ It has been shown that HA can bind to oxygen-containing donor groups to form zinc hyaluronate (Zn-HA) complexes.

[0003] Zinc hyaluronate is usually obtained by exchanging sodium ions in sodium hyaluronate for zinc ions in a zinc salt.

[0004] A Hungarian patent reports a process for preparing powdered Zn-HA by dissolving Na-HA in water, adding zinc salt, precipitating with an organic solvent, and then dehydrating and drying using an organic solvent. Currently, the ion-exchange resin method is commonly used in China, where sodium hyaluronate is dissolved and passed through an ion-exchange column to convert Na-HA to Zn-HA, which is then precipitated with ethanol, dehydrated, and dried. The commonality between the two methods is that Na-HA is first dissolved and then ion-exchanged in solution. The former method uses a simpler route but results in a higher sodium ion content in the product, while the latter requires more equipment and is more complex to operate, but results in a higher zinc ion content in the product.

[0005] Patent CN100355790C, which relates to the preparation method of zinc hyaluronate, provides a method for preparing powdered zinc hyaluronate by directly ion-exchanging Na-HA solid with zinc salt in alcohol solution to produce zinc hyaluronate.

[0006] Patent CN111647100A relates to a method for preparing high molecular weight zinc hyaluronate, and by examining preparation conditions such as pH of the replacement solution, number of replacements, washing pH, number of washings, pH of the dehydration solution, and number of dehydration times, the optimum preparation conditions were obtained. The molecular weight of the obtained zinc hyaluronate reaches over 1 million Da, the light transmittance exceeds 99.5%, and the zinc content exceeds 7.0%.

[0007] All of the above processes use multiple substitutions, which have problems such as long cycle times, high organic solvent consumption, low zinc salt consumption, and significant reduction in the molecular weight of the product.

[0008] Zinc hyaluronate is a zinc salt of hyaluronic acid, which, in addition to the moisturizing effect of hyaluronic acid, has anti-inflammatory, barrier repair, skin damage repair, skin quality improvement, discoloration and whitening effects, making it suitable for use in cosmetics. Zinc hyaluronate also has physiological functions such as lubrication, antimicrobial activity, prevention and treatment of peptic ulcers, and promotion of wound repair, making it widely used in the pharmaceutical field.

[0009] Patent CN102834417A relates to a method for preparing metal hyaluronate, a method for preparing cosmetics containing metal hyaluronate, and zinc hyaluronate and its preparation method, in which the skin is treated with tape peeling to destroy the skin's barrier function, and then an essence water with a zinc hyaluronate concentration of 0.1% is applied. Experimental results show that zinc hyaluronate has an excellent effect on repairing transepidermal water loss, effectively repairing the skin barrier, and also has an excellent effect on improving redness.

[0010] Patent CN112691049A relates to a shampoo composition containing zinc hyaluronate, which is primarily used to significantly inhibit Malassezia on the scalp, eliminate dandruff, and relieve itching. It can also be combined with other ingredients to control oil and soothe the skin.

[0011] Patent CN102961396A, which is the use of hyaluronate in the preparation of drugs for treating skin diseases, the drug combination and the preparation method thereof, provides a pharmaceutical composition (cream) containing zinc hyaluronate / or sodium hyaluronate, wherein the preparations mainly composed of hyaluronic acid metal salts, especially zinc hyaluronate, are highly effective in treating skin barrier dysfunction, and can regulate the physiological functions of the skin and restore the skin barrier function. Summary of the Invention

[0012] In view of the long-standing problems in the preparation of zinc hyaluronate, the present invention is based on a solid substitution method, and optimizes the concentration of zinc salt in the substitution liquid, the volume of the substitution liquid, the substitution time, and the number of substitutions, thereby significantly shortening the production cycle, improving the substitution efficiency, shortening the contact time between the raw material and the acidic substitution liquid, and avoiding a significant decrease in the molecular weight of the product.

[0013] In view of the problems existing in the current methods for preparing zinc hyaluronate, the present invention provides a method for preparing zinc hyaluronate quickly and efficiently, which is suitable for mass production of zinc hyaluronate.

[0014] Specifically, this application adopts the following technical solutions:

[0015] 1. Adding sodium hyaluronate to an acidic aqueous organic medium containing a zinc salt and performing a single substitution to obtain a zinc hyaluronate precipitate. A method for preparing zinc hyaluronate, comprising: 2. The preparation method according to Item 1, wherein the mass ratio of the amount of sodium hyaluronate added to the amount of zinc salt is 1:0.5 to 1:3.5, preferably 1:1 to 1:3. 3. The method according to Item 1 or 2, wherein the concentration of the organic solvent in the acidic aqueous organic medium containing the zinc salt is 55% to 95% (v / v), preferably 55% to 70% (v / v), the zinc ion concentration is 1% to 3% (w / v), and the pH of the acidic aqueous organic medium containing the zinc salt is 5.0 to 6.9. 4. The preparation method according to any one of items 1 to 3, wherein the molecular weight of the sodium hyaluronate is 1 kDa to 3,000 kDa. 5. The preparation method according to any one of items 1 to 4, wherein the substitution time is 1 to 24 hours. 6. The preparation method according to any one of items 1 to 5, further comprising washing the zinc hyaluronate precipitate with a washing solution, dehydrating the washed precipitate with a dehydrating solution, and drying to obtain zinc hyaluronate powder. 7. A low-molecular-weight zinc hyaluronate prepared by the method according to any one of items 1 to 6, preferably having a molecular weight of 1000 kDa or less (equal to or less than 1000 kDa). 8. High-molecular-weight zinc hyaluronate prepared by the method according to any one of items 1 to 6, preferably having a molecular weight of more than 1000 kDa. 9. Zinc hyaluronate having a molecular weight of 2 kDa to 2500 kDa, and having a light transmittance of 88.1% or more, preferably 99.1% or more in an aqueous solution of the zinc hyaluronate at a concentration of 0.5% by weight. 10. Use of zinc hyaluronate prepared by the method according to any one of items 1 to 8 or the zinc hyaluronate according to item 9 in moisturizing the skin, controlling skin oil, inhibiting harmful skin bacteria, antioxidant, repairing scars, inhibiting scarring, and preventing skin wound infection, Preferably, the use in inhibiting harmful skin bacteria includes use in removing acne and removing dandruff; Preferably, the use in preventing skin wound infections includes the prevention of skin wound infections caused by hydroneedles and microneedles. 11. A method for moisturizing skin, controlling skin oil, inhibiting harmful skin bacteria, antioxidant, scar repair, inhibiting scarring, or preventing skin wound infection, comprising administering to a test subject zinc hyaluronate prepared by the method according to any one of items 1 to 8 or the zinc hyaluronate according to item 9, Preferably, the suppression of harmful skin bacteria includes the elimination of acne and the elimination of dandruff; Preferably, the prevention of skin wound infections includes prevention of skin wound infections caused by hydroneedles and microneedles. 12. Use of zinc hyaluronate prepared by the method according to any one of items 1 to 8 or the zinc hyaluronate according to item 9 in a skin care product. 13. Use of zinc hyaluronate in reducing skin oiliness, preferably wherein the zinc hyaluronate is a low molecular weight zinc hyaluronate; More preferably, the molecular weight of the low-molecular-weight zinc hyaluronate is 2 kDa to 1000 kDa, preferably 5 kDa to 500 kDa. 14. Use of zinc hyaluronate in reducing the porphyrin content in sebum, preferably wherein the zinc hyaluronate is a low molecular weight zinc hyaluronate; More preferably, the molecular weight of the low-molecular-weight zinc hyaluronate is 2 kDa to 1000 kDa, preferably 5 kDa to 500 kDa. 15. Use of zinc hyaluronate in removing hydroxyl radicals on the skin surface, preferably wherein the zinc hyaluronate is a low molecular weight zinc hyaluronate; More preferably, the molecular weight of the low-molecular-weight zinc hyaluronate is 2 kDa to 1000 kDa, preferably 5 kDa to 500 kDa. 16. Use of zinc hyaluronate in scavenging superoxide anion radicals on the skin surface, wherein the zinc hyaluronate is preferably a high molecular weight zinc hyaluronate; More preferably, the molecular weight of the high-molecular-weight zinc hyaluronate is 1000 kDa to 2500 kDa, preferably 1100 kDa to 2000 kDa. [Effects of the Invention]

[0016] 1. Process Improvement: This application is based on the solid-state substitution method, and optimizes the zinc salt concentration in the substitution solution, the amount (volume) of the substitution solution, the substitution time, and the number of substitutions. This significantly shortens the production cycle, improves substitution efficiency, shortens the contact time between the raw material and the acidic substitution solution, and avoids a significant decrease in the molecular weight of the product. This application provides a rapid and efficient method for preparing zinc hyaluronate, suitable for mass production. 2. Skin Care Effects: The zinc hyaluronate prepared by this application has the effects of moisturizing, inhibiting harmful skin bacteria, repairing damage, controlling oil, antioxidant properties, and removing fine wrinkles, making it suitable for skin care. Zinc hyaluronate is weakly acidic, maintaining a mildly acidic environment for the skin (including the scalp), inhibiting the growth of several pathogenic microorganisms, resisting the invasion of various fungi, and repairing skin inflammation and damage. Furthermore, zinc hyaluronate maintains skin moisture, protects and strengthens the skin's natural protective barrier, and has a certain oil-control effect, regulating oil secretion by the sebaceous glands and maintaining a balance between moisture and oil. Therefore, zinc hyaluronate plays an important role in maintaining a healthy ecological environment for skin (including the scalp). 3. The effects of zinc hyaluronate are increasingly being discovered, and much research and application has been carried out in the pharmaceutical field. Because there is no systematic and comprehensive research on the effects of zinc hyaluronate raw materials, this application carries out a series of studies on the effects of zinc hyaluronate with different molecular weights, which has more profound implications for the application and industrial development of zinc hyaluronate. [Brief explanation of the drawings]

[0017] [Figure 1A] FIG. 1 shows the effect of zinc hyaluronate I on the stratum corneum moisture content (water content). [Figure 1B] FIG. 1 shows the effect of zinc hyaluronate II on the moisture content of the stratum corneum. [Figure 2A] FIG. 1 shows the effect of zinc hyaluronate I on skin oil amount (oil content). [Figure 2B] FIG. 1 shows the effect of zinc hyaluronate II on skin oil content. [Figure 3A] FIG. 1 shows the effect of zinc hyaluronate I on T-zone porphyrin content. [Figure 3B] FIG. 1 shows the effect of zinc hyaluronate II on T-zone porphyrin content. [Figure 4] FIG. 1 shows the hydroxyl radical scavenging effect of zinc hyaluronate. [Figure 5] FIG. 1 shows the superoxide anion radical scavenging effect of zinc hyaluronate. [Figure 6A] FIG. 1 shows changes in collagen I content. [Figure 6B] FIG. 1 shows changes in collagen III content. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present application will be further described below with reference to examples, but it should be understood that these examples are used only to further describe the present application and are not intended to limit the present application.

[0019] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In the event of a conflict, the definitions contained herein shall prevail. Furthermore, the materials, methods, and examples are illustrative only and not limiting. The present application is further described below with reference to specific embodiments, but are not intended to limit the scope of the present application.

[0020] The method for preparing zinc hyaluronate described in this application quickly and efficiently comprises: preparing an acidic aqueous-organic medium containing a zinc salt; Immersing the sodium hyaluronate solid in an acidic aqueous organic medium containing zinc salt, and stirring to replace it, and controlling the replacement time so that the zinc ion content meets the requirement; When the zinc ion content reaches the required level, the supernatant is removed and the residue is washed with an acidic aqueous organic medium to remove excess ions; After washing, the product is dehydrated and vacuum dried to obtain zinc hyaluronate.

[0021] In this application, the zinc salt refers to a salt that can at least partially dissociate in an aqueous solution to generate zinc ions.Exemplary zinc salts include, but are not limited to, zinc lactate, zinc oxide, zinc chloride, zinc phosphate, zinc citrate, zinc acetate, zinc sulfate, zinc nitrate, zinc borate, zinc butyrate, zinc carbonate, zinc formate, zinc gluconate, zinc glycerate, zinc glycolate, zinc oxide, zinc phosphate, zinc picolinate, zinc propionate, zinc salicylate, zinc silicate, zinc stearate, zinc tartrate, zinc undecenoate, and mixtures thereof.

[0022] In certain preferred embodiments, the zinc salt is zinc chloride, zinc acetate, zinc sulfate, zinc nitrate, or zinc lactate.

[0023] In the present application, the organic medium is an organic medium that has good compatibility with water but in which sodium hyaluronate or zinc hyaluronate is insoluble or hardly soluble, such as an alcohol-based organic solvent, a ketone-based organic solvent, an amide-based solvent, or acetonitrile, and an alcohol-based organic solvent or a ketone-based organic solvent is preferred.

[0024] Exemplary alcohol-based organic solvents include, but are not limited to, methanol, ethanol, isopropyl alcohol, propanol, n-butanol, diacetone alcohol, ethylene glycol ethyl ether, ethylene glycol butyl ether, propylene glycol butyl ether, propylene glycol monomethyl ether, and diethylene glycol monobutyl ether.

[0025] In certain preferred embodiments of the present application, the alcoholic organic solvents are methanol and ethanol.

[0026] Exemplary ketone solvents include, but are not limited to, methyl ethyl ketone, methyl isobutyl ketone, 1-methyl-2-pyrrolidone, cyclohexanone, or acetone.

[0027] In a particular preferred embodiment of the present application, the ketone organic solvent is acetone.

[0028] Exemplary amide solvents include, but are not limited to, N,N-dimethylacetamide, N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, or formamide.

[0029] In the present application, the acidic aqueous organic medium refers to an aqueous organic medium prepared by dissolving any of the above organic media in water to obtain a certain proportion of the aqueous organic medium, and at the same time adjusting the pH value of the aqueous organic medium to an acidic value. Examples of substances for adjusting the pH value of the aqueous organic medium include hydrochloric acid, glacial acetic acid, sulfuric acid, and phosphoric acid.

[0030] In certain preferred embodiments of the present application, the pH value of the acidic aqueous-organic medium is in the range of 5.0 to 6.9, for example, the pH value may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9.

[0031] In an acidic environment, solid sodium hyaluronate has a high degree of dissociation of sodium ions, making ion exchange easier. However, if the pH is too low, sodium hyaluronate will decompose, affecting the molecular weight of the product. The longer the product is in contact with the acidic aqueous medium, the more significantly the molecular weight will decrease. Therefore, it is important to control the appropriate pH and production cycle.

[0032] In some preferred embodiments of the present application, the mass concentration of the organic medium is 55 to 95% by weight, preferably 55 to 70% by weight.

[0033] Within this concentration range, sodium hyaluronate is insoluble and solid, so the lower the concentration of the aqueous medium, the higher the dispersion of sodium hyaluronate, but the slower the sedimentation, the greater the loss, and the longer the cycle. Conversely, the lower the dispersion, the smaller the loss, and the faster the sedimentation.

[0034] The zinc ion concentration in the acidic aqueous organic medium containing the zinc salt may be 1 to 3% by weight, for example, 1% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, 2.4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, or 3% by weight.

[0035] The concentration of zinc ions is related to the concentration of the organic solvent in the acidic aqueous organic medium; the higher the concentration of the organic solvent, the lower the solubility of zinc ions. Therefore, in an appropriate aqueous organic medium, the zinc ion concentration is preferably 1-3 wt %, which not only ensures that the zinc ion concentration reaches a certain level, but also promotes the substitution reaction well and ensures that the zinc salt is completely dissolved in the acidic aqueous organic medium.

[0036] In the present application, the sodium hyaluronate is a white or off-white solid, and the molecular weight and uronic acid content of the sodium hyaluronate can be any value. In a preferred embodiment of the present application, the molecular weight of the sodium hyaluronate is 1 kDa to 3,000 kDa, preferably 10 kDa to 2,500 kDa, and the uronic acid content is 40 to 50 wt%, preferably 45 to 50 wt%.

[0037] Due to degradation during the preparation process, the molecular weight of the sodium hyaluronate selected will be higher than the molecular weight of the zinc hyaluronate required.

[0038] The ratio of the charged mass of sodium hyaluronate to the mass of zinc salt is 1:0.5 to 1:3.5, and may be, for example, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, or 1:3.5, and is preferably 1:1 to 1:3.

[0039] The stirring and replacement time is 1 to 24 hours, preferably 5 to 16 hours.

[0040] When sodium hyaluronate undergoes a substitution reaction in an acidic aqueous organic medium containing a zinc salt, the higher the zinc ion concentration in the acidic aqueous organic medium, the greater the total amount of zinc salt used, and the longer the substitution time, the higher the zinc ion content in the product and the higher the substitution rate.The volume of the substitution solution is controlled by controlling the total amount of zinc salt according to the content of zinc salt in the acidic aqueous medium, and the substitution efficiency is high when the ratio of the charged mass of sodium hyaluronate to the mass of zinc salt is 1: (0.5-3.5).The longer the stirring time for substitution, the higher the substitution efficiency, but the longer the time, the more the product decomposes and the lower the molecular weight, so the substitution time is controlled to 1-24 hours, and more preferably, 5-16 hours.

[0041] In the above preparation method, the pH of the acidic aqueous organic medium in the washing step is 5.0 or more and less than 6.9, the concentration of the organic medium is 70 to 85% by weight, and the number of washings is 2 to 6 times.

[0042] Furthermore, after the zinc ions are substituted to meet the requirements, the product is washed to wash away excess unbound ions, and at the same time, the bound ions are more stabilized. Washing is carried out using an acidic aqueous organic medium. In the acidic aqueous organic medium, the organic medium is an organic medium that has good compatibility with water but is insoluble or slightly soluble in sodium hyaluronate or zinc hyaluronate, preferably an alcohol-based organic solvent or a ketone-based organic solvent, and ethanol, methanol, acetone, etc. are commonly used.

[0043] In the acidic aqueous organic medium, the concentration of the organic medium is 70 to 85% by weight, and the pH is 5.0 to 6.9.

[0044] Cleaning is based on the complete removal of excess ions.

[0045] To improve the cleaning effect and reduce wastewater generation, the cleaning process can be repeated multiple times.

[0046] In addition, after washing is completed, use neutral aqueous organic medium to dehydrate product, and the concentration of organic medium is more than 90% by weight.This organic medium is the organic medium that has good compatibility with water, but sodium hyaluronate or zinc hyaluronate is insoluble or hardly soluble in, preferably alcohol-based organic solvent or ketone-based organic solvent, and commonly uses ethanol, methanol, acetone, etc.

[0047] After dehydration, the aqueous organic medium in the supernatant is removed and dried to obtain zinc hyaluronate solid.

[0048] The drying method is vacuum drying, and the drying temperature can be adjusted according to the molecular weight requirements and loss on drying requirements of the product, but is generally 20 to 75°C.

[0049] Furthermore, the zinc ion content of the zinc hyaluronate obtained by the above preparation method is 6.0 to 9.0% by weight, the substitution rate is 75% to 100%, and other indicators include a pH of 5.5 to 7.5, loss on drying of 15.0% or less, light transmittance of 99.0% or more, heavy metals of 20 ppm or less, protein of 0.1% or less, zinc hyaluronate content of 90% by weight or more, and zinc hyaluronate yield of 90% or more.

[0050] Furthermore, the process for preparing zinc hyaluronate may include a step of decomposing sodium hyaluronate in addition to the steps of stirring and replacing, washing, dehydrating, and drying, and this decomposition step is carried out before stirring and replacing.

[0051] Degradation of sodium hyaluronate can be achieved by any method disclosed in the prior art, such as enzymatic hydrolysis, alkaline hydrolysis, etc.

[0052] Since sodium hyaluronate decomposes in an acidic environment, in a specific embodiment of the present application, when preparing zinc hyaluronate with a lower molecular weight, sodium hyaluronate is first decomposed in an acidic aqueous organic medium to decompose the molecular weight of sodium hyaluronate to the required molecular weight, and then the decomposed sodium hyaluronate is replaced by stirring to prepare zinc hyaluronate.

[0053] The pH during decomposition is different from the pH during stirring and replacement, and is less than 5, preferably greater than 1 and less than 5.

[0054] After completion of the decomposition, sodium hyaluronate, the pH of which has been adjusted with sodium hydroxide, is added to the zinc salt-containing acidic aqueous organic medium and stirred to replace the acidic aqueous organic medium.

[0055] In the present application, the method for preparing zinc hyaluronate is based on the existing method for preparing zinc hyaluronate, and has been further optimized through numerous experimental trials. By adjusting the mass ratio of sodium hyaluronate and zinc salt during the replacement process to an appropriate ratio during the preparation process, a good replacement effect between sodium hyaluronate and zinc salt can be achieved in a single replacement, eliminating the need for repeated addition of zinc salt for multiple replacements as in the prior art, thereby avoiding the waste of human and material resources. In the present application, the concentration of the solvent used in the preparation process and the molecular weight of sodium hyaluronate are further optimized, and the process parameters are strengthened to further ensure the quality of the prepared zinc hyaluronate. The preparation method of zinc hyaluronate of the present application simplifies the preparation steps, saves resources, and the prepared zinc hyaluronate is effective and has obvious advantages in both light transmittance and yield.

[0056] By controlling the degradation step and selecting the molecular weight of sodium hyaluronate, zinc hyaluronate with different molecular weights in the range of 1 kDa to 2000 kDa can be obtained, for example, 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 200 kDa, 200 kDa to 500 kDa, 500 kDa to 2000 kDa, etc.

[0057] Furthermore, the zinc hyaluronate prepared by the present application has the effects of moisturizing, inhibiting harmful skin bacteria, repairing damage, controlling oil, antioxidation, and removing fine wrinkles, and can maintain a healthy ecological environment for skin (including scalp), and can be used as a raw material in the field of cosmetics.

[0058] Zinc hyaluronate is a zinc salt of hyaluronic acid. It not only possesses the excellent properties of HA, but also unique physiological functions and effects. Zinc hyaluronate with different molecular weights has different biological activities. Here, low-molecular-weight zinc hyaluronate, also known as hydrolyzed zinc hyaluronate, refers to zinc hyaluronate with a molecular weight of less than 1000 kDa, obtained by the substitution reaction of hydrolyzed sodium hyaluronate with zinc ions. Zinc hyaluronate has a significant inhibitory effect on various bacteria on the skin surface (e.g., Staphylococcus epidermidis, Malassezia, and Propionibacterium acnes), and experiments have confirmed that high-molecular-weight zinc hyaluronate is more effective than hydrolyzed zinc hyaluronate. Zinc hyaluronate also has the effect of reducing epidermal oil. Porphyrins are products of microbial metabolism, and there is a certain correlation between porphyrin content and oil content in the skin. Areas with higher oil content typically have more active microbial metabolic activity and produce more porphyrins. Zinc hyaluronate can reduce porphyrin content in the T-zone to some extent, but the effect of low-molecular-weight zinc hyaluronate is more pronounced. Low-molecular-weight zinc hyaluronate can also reduce fine wrinkles, improve skin texture, and delay skin aging. Furthermore, zinc hyaluronate can significantly inhibit collagen I synthesis, thereby affecting the expression ratio of collagen I and collagen III proteins and suppressing scarring caused by overexpression of collagen I.

[0059] Furthermore, the zinc hyaluronate can be used to prepare skin care products, including moisturizing, antibacterial, anti-inflammatory, oil control, anti-aging and repairing.

[0060] Further, the skin care products are toners, essences, gels, lotions, creams, facial masks, makeup, soaps, facial cleansers, shampoos, conditioners, and body washes.

[0061] Additionally, various forms of skin care products containing zinc hyaluronate are also covered by this application.

[0062] The zinc hyaluronate prepared by the present application is used for skin moisturizing, sebum control, inhibition of harmful skin bacteria, antioxidant, scar repair, inhibition of scarring, and prevention of skin wound infection.

[0063] Through extensive experimental research, the present application has found that there is a certain correlation between the porphyrin content and oil content of the skin, and that areas with a high oil content usually have more active microbial metabolic activity and produce more porphyrin. The zinc hyaluronate prepared by the present application can significantly maintain the porphyrin content of the skin at a lower level, thereby effectively controlling the oil content of the skin. In a preferred embodiment, the molecular weight of the zinc hyaluronate is 2 kDa to 1000 kDa, which has a better oil control effect. For example, the molecular weight can be 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 120 kDa, 140 kDa, 160 kDa, 180 kDa, 190 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 400 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, 510 kDa, 520 kDa, 530 kDa kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, and more preferably 5 kDa to 500 kDa.

[0064] The zinc hyaluronate prepared in this application has excellent effects in removing acne and dandruff. Further research into this application has shown that the zinc hyaluronate exhibits a high inhibition rate against Staphylococcus epidermidis, potentially reaching 50%, and an inhibition rate against Malassezia furfur, potentially reaching 70% or more. Since Malassezia furfur is the main cause of dandruff, the zinc hyaluronate prepared in this application can be effectively used to remove dandruff. The zinc hyaluronate also exhibits an inhibition rate of 60% or more against Propionibacterium acnes, which is the main cause of skin acne. Therefore, the zinc hyaluronate prepared in this application can be effectively used to remove acne. Furthermore, this application can also prevent infection of skin wounds caused by water-light needles, microneedles, etc.

[0065] The hyaluronic acid prepared in the present application has excellent application as an antioxidant, and the zinc hyaluronate has a great effect in scavenging hydroxyl radicals, and the low molecular weight zinc hyaluronate has a higher scavenging rate of hydroxyl radicals, reaching 80% or more. In a preferred embodiment, the molecular weight of the low molecular weight zinc hyaluronate is 2 kDa to 1000 kDa to obtain better effects, for example, the molecular weight is 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa. a, 250kDa, 300kDa, 350kDa, 350kDa, 400kDa, 450kDa, 500kDa, 550kDa, 600kDa, 650kDa, 700kDa, 750kDa, 800kDa, 850kDa, 900kDa, 950kDa, 1000kDa, and more preferably 5kDa to 500kDa.

[0066] The present application further finds that the zinc hyaluronate has excellent application in scavenging superoxide anion radicals on the skin surface, and that the zinc hyaluronate has a significant effect in scavenging superoxide anion radicals, and among them, high molecular weight zinc hyaluronate has a higher scavenging ability, with the scavenging rate reaching 70% or more. In one preferred embodiment, when the molecular weight of the high molecular weight zinc hyaluronate is 1000kDa to 2500kDa, it has a better effect, for example, the molecular weight is 1100kDa, 1150kDa, 1200kDa, 1250kDa, 1300kDa, 1350kDa, 1400kDa, 1450kDa, 1500kDa, 1550kDa, 1600kDa, 1650kDa. a, 1700kDa, 1750kDa, 1800kDa, 1850kDa, 1900kDa, 1950kDa, 2000kDa, 2050kDa, 2100kDa, 2150kDa, 2200kDa, 2250kDa, 2300kDa, 2350kDa, 2400kDa, 2450kDa, 2500kDa, and more preferably 1100kDa to 2000kDa.

[0067] Beneficial effects 1. In this application, zinc hyaluronate can be prepared with only one substitution, and the zinc ion content in the prepared zinc hyaluronate is more than 4% by weight, the substitution rate is more than 56%, the light transmittance is more than 88.0%, and the yield is more than 89%. 2. The preparation of zinc hyaluronate in this application is rapid and efficient, which can greatly save production time, labor and material costs, and the obtained zinc hyaluronate has a high yield and is suitable for large-scale industrial production. [Example]

[0068] The present application will be described in detail below using examples. However, it should be understood that the present application can be implemented in various forms and should not be limited to the examples set forth herein. Rather, these examples are provided to fully understand the present application and fully convey the scope of the present application to those skilled in the art. The numerical ranges recited in this application include the endpoints of the numerical range and each specific numerical value within the numerical range, and these numerical values ​​and endpoints can be arbitrarily combined to form new subranges.

[0069] In the following examples and comparative examples, sodium hyaluronate raw materials used were all manufactured by Bloomage Biotechnology Corporation Limited.

[0070] In the following examples, the uronic acid content was determined by the sulfuric acid-carbazole colorimetric method, the sodium content and zinc content by atomic absorption spectrometry, and the molecular weight by the intrinsic viscosity method.

[0071] In the following examples, substitution rate refers to the substitution efficiency of zinc ion in zinc hyaluronate.Substitution rate is calculated as the ratio between the actual detected value of zinc ion in zinc hyaluronate and the theoretical value of complete substitution of zinc ion.

[0072] In the following examples, the yield is the ratio of the discharge amount to the charged amount, and is calculated by the following formula: Yield = Zinc hyaluronate output / Sodium hyaluronate input x 100%

[0073] In the following examples, the light transmittance of a 0.5% aqueous solution at 550 nm was measured using a spectrophotometer.

[0074] In the following examples, all concentrations given are mass percentage concentrations unless otherwise specified.

[0075] Example 1 Reagents: Ethanol, Bloomage Biotechnology Corporation Limited; Glacial acetic acid, Sinopharm Chemical Reagent Co.,Ltd.; Sodium hyaluronate, Bloomage Biotechnology Corporation Limited; Zinc acetate, Sinopharm Chemical Reagent Co.,Ltd.; Zinc chloride, Sinopharm Chemical Reagent Co.,Ltd.; Zinc sulfate, Sinopharm Chemical Reagent Co.,Ltd.

[0076] 170 L of a zinc acetate-ethanol solution containing 1.88 wt % zinc ions was prepared, in which the ethanol concentration was about 55 wt %, and the pH was adjusted to 6.5 with glacial acetic acid. 30 kg of solid sodium hyaluronate (molecular weight 2500 kDa) was accurately weighed and added to the above replacement liquid, and stirring was started to replace the liquid, followed by stirring for 8 hours. The mixture was left to stand until the supernatant became clear, then the supernatant was removed, and 800 L of a 70 wt% aqueous ethanol solution (pH 6.3) was added to wash. After washing and stirring for 3 hours, the mixture was left to stand until the supernatant became clear, the supernatant was removed, and another 800 L of a 70 wt% aqueous ethanol solution (pH 6.3) was added and washed in the same manner, for a total of four washes. After the final washing, the mixture was left to stand and the supernatant was removed, and 800 L of a 90% by weight ethanol solution was added for dehydration, and the mixture was dehydrated twice. It was then transferred to a three-in-one dryer and vacuum dried at a drying temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 18 hours, yielding 28.33 kg of zinc hyaluronate as the product.

[0077] Examples 2 to 17 The zinc hyaluronate of Examples 2 to 17 was prepared in the same manner as in Example 1. The amounts of each substance and technical parameters in the methods are shown in the table. Technical parameters not listed in the table are the same as in Example 1.

[0078] [Table 1]

[0079] Test Example 1 Product performance and quality of zinc hyaluronate The product performance and quality of the zinc hyaluronate prepared in Examples 1 to 18 were measured, and the indexes are shown in Table 2. As can be seen from the table, by adjusting the appropriate ratio and concentration of each component and the appropriate parameter index, the present application can achieve the preparation of zinc hyaluronate with only one substitution, and the zinc ion content of the prepared zinc hyaluronate is more than 4 wt%, the substitution rate is more than 56%, the light transmittance is more than 88.0%, and the yield is more than 89%, and the quality index of the product meets the requirements.

[0080] [Table 2]

[0081] Test Example 2: Study on the properties of zinc hyaluronate Test materials: Zinc hyaluronate (Example 9, molecular weight 1270 kD), sodium hyaluronate (Bloomage Biotechnology Corporation Limited, molecular weight 1230 kD)

[0082] Experimental process: Aqueous solutions of 0.5% zinc hyaluronate and 0.5% sodium hyaluronate (containing 0.13 g of zinc acetate dihydrate) were prepared so that the zinc ion content of each sample was 0.039%, and the samples were designated HA-Zn and HA-Na+Zn salt, respectively. The dynamic viscosity, light transmittance, and osmotic pressure of the two solutions were measured.

[0083] Test Results: (1)Kinematic viscosity The kinematic viscosity of a 0.5% concentration sample solution was measured at 25°C, and the results are shown in Table 3.

[0084] [Table 3]

[0085] (2) Light transmittance A spectrophotometer was used to measure the light transmittance of the samples at 550 nm, and the results are shown in Table 4.

[0086] [Table 4]

[0087] (3) Osmotic pressure The osmotic pressure of the samples was measured, with the osmotic pressure of water being set at 0. The results are shown in Table 5.

[0088] [Table 5]

[0089] The above results show that compared with sodium hyaluronate solutions containing the same zinc ion content, zinc hyaluronate has higher dynamic viscosity and light transmittance, but lower osmotic pressure.

[0090] Test example 2.1 Patch test Experimental materials Sample 1: High molecular weight zinc hyaluronate sample (Example 9, 1270 kD); Sample 2: Low molecular weight zinc hyaluronate sample (Example 10, 45 kD)

[0091] Experimental process: (1) Sample preparation Purified water was used as a control, and Sample 1 and Sample 2 were prepared in purified water to concentrations of 1.0% and 0.5%, respectively. (2) Skin patch test The patch tester package was torn open, and 0.025 mL of each prepared sample was measured and added to the chamber. The patch tester was placed on the curved surface of the subject's forearm and gently pressed with the palm of the hand to evenly blend the test material into the skin. The test was continued for 24 hours. Thirty subjects were tested. 3. Analyzing the Results The reaction results were observed and recorded according to Table 6 30 minutes, 24 hours, and 48 hours after removing the patch tester.

[0092] [Table 6]

[0093] [Table 7]

[0094] The results, as shown in Table 7, showed that all 30 subjects had negative reactions to the high and low molecular weight zinc hyaluronate samples at concentrations of 1.0% and 0.5%, indicating that there were no potential side effects on the human body.

[0095] Test Example 2.2 Moisturizing effect Test materials: Emulsion containing 0.5% zinc hyaluronate I (Example 9), emulsion containing 0.5% zinc hyaluronate II (Example 10); blank emulsion (no zinc hyaluronate). Other ingredients in the emulsion formulation are listed in Table 8 below.

[0096] [Table 8]

[0097] Experimental process: Using the half-face comparison method, the subjects were divided into two groups, A and B, with 10 subjects in each group. Group A: The emulsion containing high molecular weight zinc hyaluronate (Example 9) was used on the left face, and the blank emulsion was used on the right face; Group B: The emulsion containing low molecular weight zinc hyaluronate (Example 10) was used on the left face, and the blank emulsion was used on the right face. The moisture content of the stratum corneum in the apple muscle area of ​​the subject's face was measured before and after use.

[0098] Test Results: The initial value was set to 100% before use, and the results for Groups A and B are shown in Figure 1A and Figure 1B, respectively. Compared to the blank group, both high-molecular-weight and low-molecular-weight zinc hyaluronate were able to increase the moisture content of the stratum corneum (4%-8%) during 4 weeks of use.

[0099] Test Example 2.3: Oil Control Effect Test materials: The two emulsions prepared in Test Example 2.2

[0100] Experimental process: Using the half-face comparison method, the subjects were divided into two groups, A and B, with 10 subjects in each group. Group A: The emulsion containing high molecular weight zinc hyaluronate (Example 9) was used on the left face, and the blank emulsion was used on the right face; Group B: The emulsion containing low molecular weight zinc hyaluronate (Example 10) was used on the left face, and the blank emulsion was used on the right face. Before and after use, the subjects' forehead oil secretion, T-zone porphyrin content, and facial skin area in the apple muscle region were measured.

[0101] Test Results: 1. Effect of test sample on skin oil The results for Groups A and B are shown in Figures 2A and 2B, respectively, and are expressed as the difference in skin oil content from before use. In Figure 2A, the skin oil content of the high molecular weight zinc hyaluronate group was slightly lower than that of the blank control group, with the mean oil content at 5.70 μg / cm at week 4. 2On the other hand, in Figure 2B, the effect of reducing skin oil in the low molecular weight zinc hyaluronate group was more pronounced, especially after one week of use, the average skin oil content was 13.11 μg / cm compared to the blank group. 2 It decreased. 2. Effect of test samples on porphyrin content in the T-zone Porphyrins are products of microbial metabolism, and there is a certain correlation between porphyrin content and oil content in the skin. Areas with higher oil content typically have more active microbial metabolic activity and produce more porphyrins. The initial value was set at 100% before use, and the results for Groups A and B are shown in Figure 3A and Figure 3B, respectively. In Figure 3A, compared to the blank group, zinc hyaluronate I was able to reduce the porphyrin content in the T-zone to a certain extent (a 4%-6% reduction) only after two weeks of use. In Figure 3B, compared to the blank group, zinc hyaluronate II consistently maintained a low T-zone porphyrin content (a 7%-14% reduction) during four weeks of use.

[0102] Test Example 2.4: Suppression of harmful bacteria on the skin Test materials: Zinc Hyaluronate Sample (Example 9, 1270 kD)

[0103] Experimental process: 1) The test bacteria suspension was appropriately diluted with PBS solution to obtain the required concentration, and 0.1 mL was added dropwise to 5.0 mL of the control sample solution (PBS phosphate buffer solution). The number of recovered bacteria was 1 x 10 4 ~9×10 4 The cells were diluted to give cfu / mL. 2) The test sample was diluted to a predetermined concentration with sterilized standard hard water. 3) 5.0 mL of the test sample stock solution or its diluted solution was placed in a sterilized test tube and stored at a constant temperature of 20°C for 5 minutes. 4) 0.1 mL of the test bacteria solution was added to a test tube containing 5.0 mL of sample, quickly mixed uniformly, and the time was immediately measured. 5) After the set time, 0.5 mL of the mixture of test bacteria and sample was taken and added to a test tube containing 4.5 mL of sterilized PBS, and mixed well. 6) After leaving the plate for 10 minutes, 1 mL of the sample solution (or two or three dilutions after appropriate dilution) was placed on a sterile plate. Two sterile plates were inoculated with each sample solution or dilution. 15 mL of nutrient agar (bacteria) or Sabouraud agar (Candida albicans) cooled to 40-45°C was poured onto the plate, and the plate was rotated to ensure complete uniformity. Once the agar had solidified, the plate was turned over. After incubation at 35±2°C for 48 hours (bacteria) or 72 hours (Candida albicans), the viable colonies were counted. 7) Instead of the test sample, PBS was used as a control sample following the above procedure. 8) Calculation of antibacterial rate Antibacterial rate (%) = (average number of colonies in the control sample - average number of colonies in the test sample) / average number of colonies in the control sample × 100%

[0104] [Table 9]

[0105] The results are shown in Table 9. After 8 hours of treatment with 0.5% zinc hyaluronate solution, the inhibition rate against Staphylococcus epidermidis was 50.03%, against Malassezia furfur, 72.06%, and against Propionibacterium acnes, 62.10%. Therefore, zinc hyaluronate is effective in suppressing harmful bacteria on the skin surface and can prevent skin wound infections caused by water needles and microneedles. It can also be used in products to remove dandruff (caused by Malassezia furfur) and acne (caused by Propionibacterium acnes).

[0106] Test Example 2.5 Antioxidant effect (1) Hydroxy radical scavenging effect Experimental materials: 0.5% zinc hyaluronate I (Example 9, 1270 kD) solution sample, 0.5% zinc hyaluronate II (Example 10, 45 kD) sample; 0.5% sodium hyaluronate solution sample (Bloomage Biotechnology Corporation Limited, 1230 kD), 0.5% zinc acetate (dihydrate) solution; Experimental Method: The salicylic acid method was used to measure the hydroxyl radical scavenging ability of zinc hyaluronate samples. A certain amount of H2O2 and FeSO4 were mixed together to generate hydroxyl radicals through the Fenton reaction. Salicylic acid was then added to the reaction system to capture the hydroxyl radicals, producing 3-hydroxysalicylic acid and 5-hydroxysalicylic acid, both of which exhibit strong absorption at 510 nm. The reaction scheme is as follows: H2O2+Fe 2+ →·OH+OH - +Fe 3+ When the sample was added, it competed with salicylic acid and reacted with OH, reducing the production of 3-hydroxysalicylic acid and 5-hydroxysalicylic acid. The absorbance value of the reaction solution at 510 nm was measured using the fixed reaction time method and compared with that of a blank solution to determine the scavenging effect of the test substance on hydroxyl radicals. The erasure rate is calculated as follows: S / %=A0-(A x -A x0 ) / A0×100 where S is the quenching rate, A is the absorbance of the blank control, and A x is the absorbance upon addition of the sample, A x0 is the absorbance when no color former is added.

[0107] Test Results: The results in Figure 4 clearly show that HA-Zn has a significant effect on scavenging hydroxyl radicals, while low-molecular-weight zinc hyaluronate exhibits a higher hydroxyl radical scavenging rate, reaching 85%. At the same concentration, HA-Na and zinc acetate dihydrate alone also have a certain ability to scavenge hydroxyl radicals, but their scavenging rates are significantly lower than those of HA-Zn. Therefore, zinc hyaluronate has a significant effect on scavenging hydroxyl radicals, which indicates that zinc hyaluronate has excellent antioxidant properties and is very important for maintaining normal physiological activity and preventing aging.

[0108] (2) Superoxide anion scavenging effect Test materials: 0.5% zinc hyaluronate I (Example 9, 1270 kD) solution sample, 0.5% zinc hyaluronate II (Example 10, 45 kD) sample; 0.5% sodium hyaluronate solution sample (Bloomage Biotechnology Corporation Limited, 1230 kD), 0.5% zinc acetate (dihydrate) solution

[0109] Experimental Method: The pyrogallol autoxidation method was used. Under weakly alkaline conditions, pyrogallol undergoes autoxidation, producing superoxide anions and a colored intermediate product with a characteristic absorption peak at 320 nm. In the preliminary test, the amount of the intermediate product is linearly related to time. Because the autoxidation rate depends on the concentration of superoxide anions, the addition of a superoxide anion scavenger quickly reacts with the superoxide anions to inhibit the autoxidation reaction, preventing the accumulation of intermediate products and weakening the solution's light absorption at 320 nm. Therefore, the scavenging effect of the scavenger on superoxide anions can be evaluated by measuring the A320 value.

[0110] The erasure rate is calculated as follows: Superoxide anion radical scavenging rate (%) = (A0 - A x ) / A0×100%

[0111] As can be seen from the results in Figure 5, HA-Zn has a significant effect in scavenging superoxide anion radicals, and high-molecular-weight zinc hyaluronate exhibits a higher scavenging rate, reaching over 70%. Zinc acetate has some ability to scavenge superoxide anion radicals, but it is significantly lower than HA-Zn, while HA-Na has almost no ability to scavenge superoxide anion radicals. Therefore, zinc hyaluronate has a significant effect in scavenging superoxide anion radicals, indicating that zinc hyaluronate has excellent antioxidant effects.

[0112] Test Example 2.6 Reduction of scar formation Experimental material: Zinc hyaluronate sample (Example 9, 1270 kD)

[0113] Experimental Method: Collagen is classified into four types: I, II, III, and IV, based on the amino acid sequence of the three peptide chains that make up tropocollagen. Normal skin collagen is primarily type I and type III, with a ratio of approximately 3.5:1. During scar formation, the absolute and relative proportions of type II collagen fibers tend to decrease, while type I collagen tends to increase. Type I collagen is a thick fiber and is the material basis for scar tissue fibrosis, while type II collagen is a thin fiber and the main component of reticular fibers. As scar formation progresses, type II fibers are gradually replaced by thick type I fibers, destroying the network structure of normal skin. Accordingly, the biological characteristics of normal skin change, resulting in a significant decrease in the ratio of type I / III collagen, a disordered arrangement of collagen fibers, and thickened fibers, resulting in the characteristic appearance of scar formation.

[0114] An in vitro scar model was established by treating fibroblasts with TGF-β1. The scar model was treated with a 0.05 mg / ml zinc hyaluronate test substance working solution, and images were collected within 48 hours using a fluorescence microscope at a magnification of 400x (10x eyepiece, 40x objective). The scar repair effect was evaluated by observing changes in collagen I and collagen III content in fibroblasts. (This test was conducted by Guangdong Biocell Biotechnology Co., Ltd.)

[0115] BC is the positive control, and NC is the negative (blank) control. As can be seen from Figures 6A and 6B, after 24 hours of treatment with zinc hyaluronate at a concentration of 0.05 mg / mL, the collagen I content in the sample was significantly reduced (P<0.05), and the collagen III content was also reduced, but not significantly, compared with the NC group.

[0116] From the above, in an in vitro culture system, the sample zinc hyaluronate at a concentration of 0.05 mg / mL can significantly inhibit the synthesis of collagen I, thereby affecting the protein expression ratio of collagen I and collagen III, and inhibiting scarring caused by overexpression of collagen I.

Claims

1. adding sodium hyaluronate to an acidic aqueous organic medium containing zinc salt, and performing a single exchange to obtain a zinc hyaluronate precipitate; washing the zinc hyaluronate precipitate with a washing solution, dehydrating the washed precipitate with a dehydrating solution, and drying to obtain zinc hyaluronate powder; Including, the mass ratio of the amount of sodium hyaluronate added to the amount of zinc salt is 1:1 to 1:3; A method for preparing zinc hyaluronate, wherein the concentration of zinc ions in the acidic aqueous organic medium containing the zinc salt is 1% to 3% (w / v).

2. 2. The method according to claim 1, wherein the concentration of the organic solvent in the acidic aqueous organic medium containing the zinc salt is 55% to 95% (v / v).

3. A preparation method described in claim 1 or 2, wherein the concentration of the organic solvent in the acidic aqueous organic medium containing the zinc salt is 55% to 70% (v / v).

4. A preparation method described in claim 1 or 2, wherein the pH of the acidic aqueous organic medium containing the zinc salt is 5.0 to 6.

9.

5. 3. The method according to claim 1, wherein the molecular weight of the sodium hyaluronate is 1 kDa to 3,000 kDa.

6. The preparation method according to claim 1 or 2, wherein the substitution time is 1 to 24 hours.

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