Preparation method and application of zinc hyaluronate

Abstract

The present invention provides a method for preparing zinc hyaluronate, comprising the following steps: adding sodium hyaluronate into an acidic aqueous organic medium containing a zinc salt for a first replacement to obtain a 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. Based on the solid replacement method, the present invention optimizes the zinc salt concentration, the volume of the replacement solution, the replacement time, and the number of replacement times in the replacement solution, solves the problems of multiple replacement times, long time consumption, large molecular weight consumption, and low zinc salt utilization rate in the current zinc hyaluronate production process, greatly shortens the production cycle, improves the replacement efficiency, reduces the contact time between the raw materials and the acidic replacement solution, and avoids a significant reduction in the molecular weight of the product. The present invention provides a method for rapidly and efficiently producing zinc hyaluronate, which is applicable to the large-scale production of zinc hyaluronate.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of zinc hyaluronate, belonging to the technical field of hyaluronates. Background Art

[0002] Hyaluronic acid (HA) is a polysaccharide composed of disaccharide units of glucuronic acid and N-acetylglucosamine, and has physiological functions such as moisturizing, nourishing, repairing and preventing damage, and is widely used in the cosmetic field. Research shows that in an aqueous solution with a pH value of 6.0 - 6.5, Zn 2+ can combine with the oxygen-containing donor groups in HA to form a zinc hyaluronate (Zn-HA) complex.

[0003] Generally, zinc hyaluronate is obtained by exchanging sodium ions in sodium hyaluronate with zinc ions in zinc salts.

[0004] Hungarian patents reported the preparation process of powdered Zn-HA: dissolving Na-HA in water, adding zinc salts and then precipitating with organic solvents, and then dehydrating and drying with organic solvents. Currently, the ion exchange resin method is commonly used in China: dissolving sodium hyaluronate and loading it onto an ion exchange column to convert Na-HA into Zn-HA, and then precipitating with ethanol and dehydrating and drying. The common point of the two is to first dissolve Na-HA and perform ion exchange in the solution. The former path is relatively simple, but the sodium ion content in the product is relatively high; the latter occupies more equipment and the operation is more complex, but the zinc ion content in the product is relatively high.

[0005] In the patent CN100355790C, a preparation method of zinc hyaluronate provides a preparation method of powdered zinc hyaluronate, which directly generates zinc hyaluronate by ion exchange of solid Na-HA with zinc salts in an alcohol solution.

[0006] The patent CN111647100A, a preparation method of high molecular weight zinc hyaluronate, explores the preparation conditions such as the pH of the replacement solution, the number of replacement times, the pH of washing, the number of washing times, the pH of the dehydrating solution, and the number of dehydrating times, and obtains the optimal preparation conditions. The molecular weight of the obtained zinc hyaluronate can reach more than 1 million Da, the light transmittance is greater than 99.5%, and the zinc content is greater than 7.0%.

[0007] The above processes all adopt multiple replacements, and there are problems such as long cycle, large consumption of organic solvents, low utilization rate of zinc salts, and large reduction in the molecular weight of the product.

[0008] Zinc hyaluronate is the zinc salt of hyaluronic acid. In addition to the moisturizing properties of hyaluronic acid, it also has the following effects: anti-inflammatory, barrier repair, skin damage repair, skin quality improvement, melatonin reduction and whitening, etc., and can be used in the cosmetics field. In addition, zinc hyaluronate also has physiological functions such as lubrication, anti-microbial effect, prevention and treatment of peptic ulcers, and promotion of wound repair, and is widely used in the pharmaceutical field.

[0009] In Patent CN102834417A, a method for manufacturing a metal salt of hyaluronic acid, a method for manufacturing a cosmetic containing a metal salt of hyaluronic acid, and zinc hyaluronate and its manufacturing method, the skin barrier function is damaged by treating the skin by the tape stripping method, and essence water containing 0.1% concentration of zinc hyaluronate is applied. From the experimental results, it can be seen that zinc hyaluronate has an excellent recovery effect on the trans-epidermal water loss, and can effectively repair the skin barrier. At the same time, it has a good improvement effect on skin redness.

[0010] Patent CN112691049A discloses a shampoo composition containing zinc hyaluronate, in which zinc hyaluronate is added. The obvious inhibitory effect of zinc hyaluronate on Malassezia scalp is mainly utilized to achieve the effects of anti-dandruff and anti-itching. And it is used in combination with other ingredients to achieve the effects of oil control and soothing.

[0011] Patent CN102961396A discloses the application of hyaluronate in the preparation of drugs for treating skin diseases, its drug combination and its preparation method, and provides a drug composition (cream) containing zinc hyaluronate / or sodium hyaluronate. The preparation with the metal salt of hyaluronic acid, especially zinc hyaluronate as the main active ingredient, has a definite curative effect on treating skin barrier function disorders, and can regulate skin physiological functions and repair skin barrier functions. Summary of the Invention

[0012] Aiming at the long-term existing problems in the production of zinc hyaluronate, based on the solid displacement method, the present invention optimizes the zinc salt concentration, displacement liquid volume, displacement time, and displacement times in the displacement liquid, greatly shortening the production cycle, improving the displacement efficiency, reducing the contact time between the raw material and the acidic displacement liquid, and avoiding a significant reduction in the molecular weight of the product.

[0013] Aiming at the problems existing in the current preparation methods of zinc hyaluronate, the present invention provides a method for rapidly and efficiently producing zinc hyaluronate, which is suitable for the large-scale production of zinc hyaluronate.

[0014] Specifically, the present invention adopts the following technical solutions:

[0015] 1. A method for preparing zinc hyaluronate, comprising the following steps:

[0016] Sodium hyaluronate is added to an acidic aqueous organic medium containing a zinc salt, and a single replacement is carried out to obtain zinc hyaluronate precipitate;

[0017] The precipitate is washed with a washing solution;

[0018] The washed precipitate is dehydrated with a dehydrating solution and dried to obtain zinc hyaluronate powder.

[0019] 2. According to the preparation method described in claim 1, the mass ratio of the addition amount of sodium hyaluronate to the zinc salt is 1:0.5 - 1:3.5, preferably 1:1 - 1:3.

[0020] 3. According to the preparation method described in item 1 or 2, the concentration of the organic solvent in the acidic aqueous organic medium containing the zinc salt is 55% - 95% (v / v), preferably 55% - 70% (v / v), the zinc ion concentration is 1% - 3% (w / v), and the pH of the acidic aqueous organic medium containing the zinc salt is 5.0 - 6.9.

[0021] 4. According to the preparation method described in any one of items 1 - 3, the molecular weight of the sodium hyaluronate is 1 kDa - 3000 kDa.

[0022] 5. According to the preparation method described in any one of items 1 - 4, the replacement time is 1 - 24 h.

[0023] 6. According to the preparation method described in any one of items 1 - 5, the concentration of the organic solvent in the washing solution is 70% - 85% (v / v), the pH of the washing solution is 5.0 - 6.9, the number of washing times is 2 - 6 times, the concentration of the organic solvent in the dehydrating solution is greater than or equal to 90% (v / v), and the number of dehydration times is more than 2 times.

[0024] 7. Zinc hyaluronate with a low molecular weight prepared by the method described in any one of items 1 - 6, preferably, the molecular weight of the low molecular weight zinc hyaluronate is less than or equal to 1000 kDa.

[0025] 8. Zinc hyaluronate with a high molecular weight prepared by the method described in any one of items 1 - 6, preferably, the molecular weight of the high molecular weight zinc hyaluronate is greater than 1000 kDa.

[0026] 9. Use of zinc hyaluronate prepared by the method described in any one of items 1 - 8 in skin moisturizing, skin oil control, inhibiting skin harmful bacteria, antioxidant, scar repair, inhibiting scarring, preventing skin wound infection,

[0027] Preferably, the use in inhibiting skin harmful bacteria includes uses in acne treatment and anti-dandruff;

[0028] Preferably, the use in preventing skin wound infection includes preventing skin wound infections caused by hydrodermabrasion needles and microneedles.

[0029] 10. Use of zinc hyaluronate prepared by the method according to any one of items 1-8 in skin care products, including scalp care.

[0030] 11. Use of zinc hyaluronate in reducing skin oiliness. Preferably, the zinc hyaluronate is low molecular weight zinc hyaluronate.

[0031] More preferably, the molecular weight of the low molecular weight zinc hyaluronate is 2 kDa - 1000 kDa, preferably 5 kDa - 500 kDa.

[0032] 12. Use of zinc hyaluronate in reducing the porphyrin content in skin oiliness. Preferably, the zinc hyaluronate is low molecular weight zinc hyaluronate.

[0033] More preferably, the molecular weight of the low molecular weight zinc hyaluronate is 2 kDa - 1000 kDa, preferably 5 kDa - 500 kDa.

[0034] 13. Use of zinc hyaluronate in scavenging hydroxyl free radicals on the skin surface. Preferably, the zinc hyaluronate is low molecular weight zinc hyaluronate.

[0035] More preferably, the molecular weight of the low molecular weight zinc hyaluronate is 2 kDa - 1000 kDa, preferably 5 kDa - 500 kDa.

[0036] 14. Use of zinc hyaluronate in scavenging superoxide anion free radicals on the skin surface. Preferably, the zinc hyaluronate is high molecular weight zinc hyaluronate.

[0037] More preferably, the molecular weight of the high molecular weight zinc hyaluronate is 1000 kDa - 2500 kDa, preferably 1100 kDa - 2000 kDa.

[0038] Effect of the invention

[0039] 1. Process improvement: Based on the solid displacement method, the present invention optimizes the zinc salt concentration, displacement liquid volume, displacement time, and displacement times in the displacement liquid, greatly shortening the production cycle, improving the displacement efficiency, reducing the contact time between the raw materials and the acidic displacement liquid, and avoiding a significant decrease in the molecular weight of the product. The present invention provides a method for rapidly and efficiently producing zinc hyaluronate, which is suitable for large-scale production of zinc hyaluronate.

[0040] 2. Skin care efficacy: The zinc hyaluronate prepared by the present invention has the effects of moisturizing, inhibiting harmful skin bacteria, repairing damage, controlling oil, antioxidation, removing fine lines, etc., and can be used for skin care. Zinc hyaluronate is weakly acidic, which can ensure the weakly acidic environment of the skin (including the scalp), and at the same time can inhibit the growth of some pathogenic microorganisms, resist the invasion of various bacteria on the skin, and repair skin inflammation and damage. In addition, zinc hyaluronate can maintain skin moisture, protect and enhance the skin's natural protection barrier, has a certain oil control effect, regulates the secretion of sebum by sebaceous glands, and maintains the water-oil balance. Therefore, zinc hyaluronate plays an important role in maintaining a healthy skin (including scalp) ecological environment.

[0041] 3. The efficacy of zinc hyaluronate has been increasingly discovered, but there are more researches and applications in the medical field. There is no systematic and comprehensive research on the role of zinc hyaluronate raw materials. Therefore, the present invention conducts a series of researches on the efficacy of zinc hyaluronate with different molecular weights, which has more profound significance for the application of zinc hyaluronate and the development of the industry. Description of the Drawings

[0042] Figure 1A Effect of zinc hyaluronate I on the water content of the stratum corneum;

[0043] Figure 1B Effect of zinc hyaluronate II on the water content of the stratum corneum;

[0044] Figure 2A Effect of zinc hyaluronate I on the skin oil content;

[0045] Figure 2B Effect of zinc hyaluronate II on the skin oil content;

[0046] Figure 3A Effect of zinc hyaluronate I on the porphyrin content in the T-zone;

[0047] Figure 3B Effect of zinc hyaluronate II on the porphyrin content in the T-zone;

[0048] Figure 4 Scavenging effect of zinc hyaluronate on hydroxyl radicals;

[0049] Figure 5 Scavenging effect of zinc hyaluronate on superoxide anion radicals;

[0050] Figure 6A Change in the content of collagen I;

[0051] Figure 6B Change in the content of collagen III. Detailed Embodiments

[0052] The present invention will be further described below in conjunction with embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present invention, and are not used to limit the present invention.

[0053] Unless otherwise defined, the technical and scientific terms in this specification 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 experiments or practical applications, the materials and methods are described below. In case of conflict, the present specification, including its definitions, shall prevail. In addition, the materials, methods, and examples are for illustrative purposes only and are not limiting. The present invention will be further described below in conjunction with specific embodiments, but not to limit the scope of the present invention.

[0054] The method for rapidly and efficiently producing zinc hyaluronate according to the present invention includes the following steps:

[0055] Prepare an acidic aqueous organic medium containing a zinc salt;

[0056] Soak the solid sodium hyaluronate in the acidic aqueous organic medium containing a zinc salt, and perform stirring and displacement. Control the displacement time to make the zinc ion content meet the requirements;

[0057] After the zinc ion content meets the requirements, add an acidic aqueous organic medium to the residue after removing the supernatant for washing to wash away the excess ions;

[0058] After washing, dehydrate the product, and then perform vacuum drying to obtain zinc hyaluronate.

[0059] In the present invention, the zinc salt refers to a salt that can at least partially dissociate in an aqueous solution to produce zinc ions. Exemplary zinc salts can 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 undecylenate, and mixtures thereof.

[0060] In some preferred embodiments, the zinc salt is zinc chloride, zinc acetate, zinc sulfate, zinc nitrate, or zinc lactate.

[0061] In the present invention, the organic medium is an organic medium that is highly compatible with water, but in which sodium hyaluronate or zinc hyaluronate is insoluble or slightly soluble, such as alcohol organic solvents, ketone organic solvents, amide solvents, or acetonitrile, preferably alcohol organic solvents or ketone organic solvents.

[0062] Exemplary alcohol organic solvents include, but are not limited to, methanol, ethanol, isopropanol, propanol, n-butanol, diacetone alcohol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, propylene glycol monobutyl ether, propylene glycol monomethyl ether, diethylene glycol monobutyl ether, etc.

[0063] In certain preferred embodiments of the present invention, the alcohol organic solvent is methanol and ethanol.

[0064] Exemplary ketone solvents include, but are not limited to, methyl ethyl ketone, methyl isobutyl ketone, 1-methyl-2-pyrrolidone, cyclohexanone, or acetone, etc.

[0065] In certain preferred embodiments of the present invention, the ketone organic solvent is acetone.

[0066] Exemplary amide solvents include, but are not limited to, N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, or formamide, etc.

[0067] In the present invention, the acidic aqueous organic medium refers to any of the above-mentioned organic media dissolved in water to form an aqueous organic medium in a certain proportion, and at the same time, the pH value of the aqueous organic medium is adjusted to be acidic. The substances for adjusting the pH value of the aqueous organic medium can be hydrochloric acid, glacial acetic acid, sulfuric acid, phosphoric acid, etc.

[0068] In certain preferred embodiments of the present invention, the pH value range of the acidic aqueous organic medium is 5.0 - 6.9. For example, the pH value can 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.

[0069] In an acidic environment, the degree of dissociation of sodium ions in sodium hyaluronate solid is high, and ion exchange is more likely to occur. However, if the pH is too low, sodium hyaluronate will be degraded, affecting the product molecular weight; and the longer the contact time between the product and the acidic aqueous medium, the more the molecular weight will decrease. Therefore, it is very important to control the appropriate pH and production cycle.

[0070] In certain preferred embodiments of the present invention, the mass concentration of the organic medium is 55 - 95 wt%, preferably 55 - 70 wt%.

[0071] Within this concentration range, sodium hyaluronate is insoluble in it and exists in a solid form. The lower the concentration of the aqueous medium, the higher the dispersion degree of sodium hyaluronate in it, but the sedimentation is slow, the loss is large, and the cycle is long; on the contrary, the dispersion degree is low, the loss is small, and the sedimentation is fast.

[0072] The zinc ion concentration in the acidic aqueous organic medium containing zinc salt is 1-3 wt%, for example, it can be 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3 wt%.

[0073] The concentration of zinc ions is related to the concentration of the organic medium in the acidic aqueous organic medium. The higher the concentration of the organic medium, the lower the solubility of zinc ions. Therefore, at a suitable concentration of the aqueous organic medium, the zinc ion concentration is preferably 1-3 wt%. This concentration can not only ensure that the zinc ion concentration reaches a certain level, but also well promote the displacement reaction and ensure that the zinc salt completely dissolves in the acidic aqueous organic medium.

[0074] In the present invention, 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 invention, the molecular weight of the sodium hyaluronate is 1 kDa-3000 kDa, preferably 10 kDa-2500 kDa, and the uronic acid content is 40-50 wt%, preferably 45-50 wt%.

[0075] Because there is degradation during the preparation process, the molecular weight of the selected sodium hyaluronate should be higher than the molecular weight of the desired zinc hyaluronate.

[0076] The mass ratio of the sodium hyaluronate feed to the zinc salt mass is 1:0.5-1:3.5, for example, it can be 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, 1:3.5, and preferably 1:1-1:3.

[0077] The stirring and displacement time is 1-24 h, preferably 5-16 h.

[0078] Perform a displacement reaction of sodium hyaluronate in an acidic aqueous organic medium containing a zinc salt. The higher the zinc ion concentration, the greater the total amount of zinc salt used, and the longer the displacement time in the acidic aqueous organic medium, the higher the zinc ion content in the product, that is, the higher the displacement rate. According to the content of the zinc salt in the acidic aqueous medium, control the volume of the displacement solution by controlling the total amount of the zinc salt. When the mass ratio of the sodium hyaluronate feed to the zinc salt mass is 1:(0.5 - 3.5), the displacement efficiency is relatively high. The longer the stirring time of the displacement, the higher the displacement efficiency, but the longer the time, the more the product degrades and the more the molecular weight decreases. Therefore, the displacement time is controlled within 1 - 24 h, and more preferably, the displacement time is within 5 - 16 h.

[0079] Further, in the above preparation method, the pH of the acidic aqueous organic medium during the washing process is greater than or equal to 5.0 and less than 6.9, the concentration of the organic medium is 70 - 85 wt%, and the number of washing times is 2 - 6 times.

[0080] Further, after the displacement reaches the requirement of zinc ions, wash the product to remove excess unbound ions and at the same time make the bound ions more stable. Wash with an acidic aqueous organic medium. In the acidic aqueous organic medium, the organic medium is an organic medium that is compatible with water but in which sodium hyaluronate or zinc hyaluronate is insoluble or slightly soluble, preferably an alcohol organic solvent or a ketone organic solvent, and commonly used ones are ethanol, methanol, acetone, etc.

[0081] In the acidic aqueous organic medium, the concentration of the organic medium is 70 - 85 wt%, and the pH is 5.0 - 6.9.

[0082] The washing is based on the standard of completely removing excess ions.

[0083] To improve the washing effect and reduce the generation of wastewater, washing can be carried out by means of multiple washings, stirring and soaking, etc.

[0084] Further, after the washing is completed, dehydrate the product. Use a neutral aqueous organic medium for dehydration, and the concentration of the organic medium is greater than or equal to 90 wt%; the organic medium is an organic medium that is compatible with water but in which sodium hyaluronate or zinc hyaluronate is insoluble or slightly soluble, preferably an alcohol organic solvent or a ketone organic solvent, and commonly used ones are ethanol, methanol, acetone, etc.

[0085] After dehydration, remove the aqueous organic medium in the supernatant and perform drying to obtain a zinc hyaluronate solid.

[0086] The drying method is vacuum drying, and the drying temperature can be adjusted according to the requirements of the molecular weight of the product and the drying loss requirements, generally 20 - 75 °C.

[0087] Furthermore, the zinc ion content of the zinc hyaluronate obtained by the above preparation method is 6.0-9.0 wt%, the replacement rate is 75%-100%, and the remaining indicators are: pH 5.5-7.5; loss on drying ≤ 15.0%, light transmittance ≥ 99.0%, heavy metals ≤ 20 ppm, protein ≤ 0.1%, the zinc hyaluronate content is more than 90 wt%, and the yield of zinc hyaluronate is more than 90%.

[0088] Furthermore, in the preparation process of zinc hyaluronate, in addition to the steps of stirring replacement, washing, dehydration, and drying, it may also include the step of degrading sodium hyaluronate, and this degradation step is carried out before stirring replacement.

[0089] Any method disclosed in the prior art can be used to achieve the degradation of sodium hyaluronate, such as enzymatic hydrolysis method, alkaline hydrolysis method, etc.

[0090] Because sodium hyaluronate will degrade in an acidic environment, in a specific embodiment of the present invention, when preparing zinc hyaluronate with a lower molecular weight, sodium hyaluronate is first degraded in an acidic aqueous organic medium to degrade the molecular weight of sodium hyaluronate to the required molecular weight, and then the degraded sodium hyaluronate is subjected to stirring replacement to prepare zinc hyaluronate.

[0091] The pH of degradation is different from the pH of stirring replacement. The pH of degradation is less than 5, preferably the pH is greater than 1 and less than 5.

[0092] After the degradation is completed, sodium hydroxide is used to adjust the pH, and then the sodium hyaluronate is added to an acidic aqueous organic medium containing a zinc salt for stirring replacement.

[0093] In this application, the preparation method of zinc hyaluronate is optimized further through multiple experimental attempts on the basis of the existing preparation method of zinc hyaluronate. By adjusting the mass ratio of sodium hyaluronate and zinc salt replacement process to a suitable ratio during the preparation process, good replacement effect can be achieved with only one replacement of sodium hyaluronate and zinc salt, avoiding the need to repeatedly add zinc salt for multiple replacements in the prior art, which consumes manpower and material resources. This application further optimizes the concentration of the solvent used in the preparation process and the molecular weight of sodium hyaluronate, strengthens the process parameters, and better ensures the quality of the prepared zinc hyaluronate. The preparation method of zinc hyaluronate in this application simplifies the preparation steps, saves resources, and the prepared zinc hyaluronate has good effects, and both the light transmittance and the yield have obvious advantages.

[0094] By regulating the degradation step and selecting the molecular weight of sodium hyaluronate, zinc hyaluronates with different molecular weight ranges within 1 kDa to 2000 kDa can be obtained, such as 1 kDa - 5 kDa, 5 kDa - 10 kDa, 10 kDa - 200 kDa, 200 kDa - 500 kDa, 500 kDa - 1000 kDa, 1000 kDa - 2000 kDa.

[0095] Furthermore, the zinc hyaluronate prepared by the present invention has functions such as moisturizing, inhibiting harmful skin bacteria, repairing damage, controlling oil, antioxidant, removing fine lines, etc., and maintains a healthy skin (including scalp) ecological environment, and can be used as a raw material in the cosmetics field.

[0096] Zinc hyaluronate is the zinc salt of hyaluronic acid, which not only has the excellent properties of HA, but also has unique physiological functions and effects, and zinc hyaluronates with different molecular weights have different biological activities. Among them, low-molecular-weight zinc hyaluronate, also called hydrolyzed zinc hyaluronate, refers to zinc hyaluronate with a molecular weight lower than 1000 kDa, which is obtained by the substitution reaction of hydrolyzed sodium hyaluronate and zinc ions. It has been experimentally verified that zinc hyaluronate has obvious inhibitory effects on various skin surface bacteria (such as Staphylococcus epidermidis, Malassezia, Propionibacterium acnes, etc.), and high-molecular-weight zinc hyaluronate has a better effect than hydrolyzed zinc hyaluronate. Zinc hyaluronate also has the effect of reducing the oil on the skin surface. Porphyrin is a metabolite of microorganisms, and there is a certain correlation between the skin porphyrin content and the oil content. In areas with higher oil content, the microbial metabolic activity is usually more vigorous, and the amount of porphyrin produced is more; zinc hyaluronate can reduce the porphyrin content in the T area to a certain extent, and low-molecular-weight zinc hyaluronate has a more significant effect. In addition, low-molecular-weight zinc hyaluronate can also reduce fine lines, improve skin texture, and delay skin aging. In addition, zinc hyaluronate can significantly inhibit the synthesis of Collagen I, thereby affecting the expression ratio of Collagen I and Collagen III proteins and inhibiting the occurrence of scars caused by the overexpression of Collagen I.

[0097] Furthermore, the zinc hyaluronate can be used to prepare skin care products. The skin care includes moisturizing, antibacterial, anti-inflammatory, oil control, anti-aging, and repair.

[0098] Furthermore, the skin care products are toner, essence, gel, lotion, cream, mask, color cosmetics, soap, facial cleanser, shampoo, hair conditioner, and body wash.

[0099] Furthermore, skin care products in various dosage forms containing zinc hyaluronate are within the scope of protection of the present invention.

[0100] Use of zinc hyaluronate prepared in this application in aspects of skin moisturizing, skin oil control, inhibition of harmful skin bacteria, antioxidant, scar repair, inhibition of scarring, and prevention of skin wound infection.

[0101] Through a large number of experimental studies in this application, it is found that there is a certain correlation between skin porphyrin content and oil content. In areas with higher oil content, the microbial metabolic activity is usually more vigorous, and the amount of porphyrin produced is more. And the zinc hyaluronate prepared in this application can significantly maintain the skin porphyrin content at a relatively low level, thereby effectively controlling the skin oil content. In a preferred embodiment, the zinc hyaluronate has a better oil control effect when its molecular weight is between 2 kDa and 1000 kDa, and is further preferably 5 kDa to 500 kDa.

[0102] The zinc hyaluronate prepared in this application has a good effect in treating acne and removing dandruff; further studies in this application find that the zinc hyaluronate has a relatively high inhibition rate against Staphylococcus epidermidis, which can reach about 50%, and at the same time, the inhibition rate against Malassezia furfur can reach more than 70%. Malassezia furfur mainly causes problems such as dandruff and skin flakes. Therefore, the zinc hyaluronate in this application has a good use in removing dandruff; the zinc hyaluronate also has an inhibition rate against Propionibacterium acnes of more than 60%, and Propionibacterium acnes mainly causes skin acne problems. Therefore, the zinc hyaluronate in this application also has a good use in treating acne. Further, this application can also prevent the infection of skin wounds caused by hydrodermabrasion needles, microneedles, etc.

[0103] The hyaluronic acid prepared in this application has a good use in antioxidant. The zinc hyaluronate has an obvious effect on scavenging hydroxyl radicals, and the hydroxyl radical scavenging rate of low-molecular-weight zinc hyaluronate is higher, which can reach more than 80%. In a preferred embodiment, the zinc hyaluronate with low molecular weight has a better effect when its molecular weight is between 2 kDa and 1000 kDa, and is further preferably 5 kDa to 500 kDa.

[0104] This application further finds that the zinc hyaluronate has a good use in scavenging superoxide anion radicals on the skin surface. The zinc hyaluronate has a significant effect on scavenging superoxide anion radicals, and the high-molecular-weight zinc hyaluronate has a higher scavenging ability, and the scavenging rate can reach more than 70%. In a preferred embodiment, the zinc hyaluronate with high molecular weight has a better effect when its molecular weight is between 1000 kDa and 2500 kDa, and is further preferably 1100 kDa to 2000 kDa.

[0105] Beneficial effects

[0106] 1. In the present invention, the preparation of zinc hyaluronate can be achieved through only one replacement, and the content of zinc ions in the prepared zinc hyaluronate is greater than 4 wt%, the replacement rate is greater than 56%, the light transmittance is greater than 88.0%, and the yield is more than 89%.

[0107] 2. The preparation of zinc hyaluronate in the present invention is rapid and efficient, saving a large amount of production time and reducing the costs of labor and materials. The obtained zinc hyaluronate has a high yield and is suitable for large-scale industrial production.

[0108] Examples

[0109] The present invention will be described in detail below with reference to examples. However, it should be understood that the present invention can be implemented in various forms and should not be limited by the examples set forth herein. On the contrary, these examples are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. In the present invention, the numerical ranges recited include the data at both ends of the numerical range, as well as each specific numerical value within the numerical range, and the numerical values can be arbitrarily combined with the endpoints to form new small ranges.

[0110] In the following examples and comparative examples, the sodium hyaluronate raw materials used are all from Bloomage Biotechnology Co., Ltd.

[0111] In the following examples, the uronic acid content was detected by the sulfuric acid-carbazole colorimetric method, the sodium content and zinc content were detected by atomic absorption spectrophotometry, and the molecular weight was detected by the intrinsic viscosity method.

[0112] In the following examples, the replacement rate refers to the replacement efficiency of zinc ions in zinc hyaluronate, and the calculation method of the replacement rate is: the ratio of the actual detected value of zinc ions in zinc hyaluronate to the theoretical value of all replaced zinc ions.

[0113] In the following examples, the yield is the ratio of the discharge amount to the feeding amount, and the calculation formula is:

[0114] Yield = discharge amount of zinc hyaluronate / feeding amount of sodium hyaluronate × 100%

[0115] In the following examples, the light transmittance of a 0.5% aqueous solution was measured at 550 nm using a spectrophotometer.

[0116] In the following examples, unless otherwise specified, the concentrations are all mass percentages.

[0117] Example 1

[0118] Reagents: ethanol, Bloomage Biotechnology Co., Ltd.;

[0119] Glacial acetic acid, Sinopharm Chemical Reagent Co., Ltd.;

[0120] Sodium hyaluronate, Bloomage Biotechnology Co., Ltd.;

[0121] Zinc acetate, Sinopharm Chemical Reagent Co., Ltd.;

[0122] Zinc chloride, Sinopharm Chemical Reagent Co., Ltd.;

[0123] Zinc sulfate, Sinopharm Chemical Reagent Co., Ltd.

[0124] Prepare 170 L of zinc acetate - ethanol solution containing 1.88 wt% zinc ions, where the ethanol concentration is approximately 55 wt%, and adjust the pH to 6.5 with glacial acetic acid.

[0125] Accurately weigh 30 kg of solid sodium hyaluronate (molecular weight 2500 kDa), add it to the above replacement solution, start stirring for replacement, and stir and replace for 8 h.

[0126] Let it stand until the supernatant is clear, remove the supernatant, add 800 L of ethanol aqueous solution with a concentration of 70 wt% (pH 6.3) for washing, wash and stir for 3 h, then let it stand until the supernatant is clear, remove the supernatant, and then add 800 L of ethanol aqueous solution with a concentration of 70 wt% (pH 6.3) to wash in the same way, and wash a total of 4 times.

[0127] After the last washing, let it stand to remove the supernatant, add 800 L of ethanol solution with a concentration of 90 wt% for dehydration operation, and dehydrate 2 times.

[0128] Then transfer it to a three - in - one dryer for vacuum drying. The drying temperature is 45 °C, the vacuum degree is 0.10 MPa, the drying time is 18 h, and 28.33 kg of zinc hyaluronate is obtained after discharging.

[0129] Example 2 - 17

[0130] Prepare zinc hyaluronate of Examples 2 - 17 according to the method of Example 1. The amounts of each substance and technical parameters in the method are shown in the table. The technical parameters not mentioned in the table are the same as those in Example 1.

[0131] Table 1 Contents and technical parameters of substances in each example

[0132]

[0133]

[0134] Test Example 1 Product performance and quality of zinc hyaluronate

[0135] The product properties and quality of the zinc hyaluronate prepared in Preparation Examples 1-18 were measured, and the various indexes are shown in Table 2. As can be seen from the table, by adjusting the reasonable ratios and concentrations of the various components and the appropriate parameter indexes, the preparation of zinc hyaluronate can be achieved only through one replacement in the present invention, and the content of zinc ions in the prepared zinc hyaluronate is greater than 4 wt%, the replacement rate is greater than 56%, the light transmittance is greater than 88.0%, the yield is more than 89%, and the quality indexes of the product meet the requirements.

[0136]

[0137]

[0138] Experimental Example 2 Study on the Properties of Zinc Hyaluronate

[0139] Experimental materials:

[0140] Zinc hyaluronate (Example 9, molecular weight 1270 kD), sodium hyaluronate (Bloomage Biotechnology Co., Ltd., molecular weight 1230 kD)

[0141] Experimental procedure:

[0142] An aqueous solution of 0.5% concentration zinc hyaluronate and 0.5% concentration sodium hyaluronate (containing 0.13 g zinc acetate dihydrate) was prepared to ensure that the zinc ion content in both samples was 0.039%. The sample names were HA-Zn and HA-Na+Zn salt respectively. The dynamic viscosity, light transmittance and osmotic pressure of the two solutions were detected.

[0143] Experimental results:

[0144] (1) Dynamic viscosity

[0145] The dynamic viscosity of the 0.5% concentration sample solution was measured at 25 °C, and the results are shown in Table 3.

[0146] Table 3

[0147] Sample name HA-Zn HA-Na + Zn salt Dynamic viscosity (mPa·s) 310.0 260.0

[0148] (2) Light transmittance

[0149] The light transmittance of the samples was measured at 550 nm with a spectrophotometer, and the results are shown in Table 4.

[0150] Table 4

[0151] Sample name HA-Zn HA-Na + Zn salt Transmittance (%) 94.62％ 81.26％

[0152] (3) Osmotic pressure

[0153] Taking the osmotic pressure of water as 0, the osmotic pressure of the samples was measured, and the results are shown in Table 5.

[0154] Table 5

[0155] Sample name HA-Zn HA-Na + Zn salt Osmotic pressure (mOsm / kg) +10 +29

[0156] As can be seen from the above results, compared with the sodium hyaluronate solution containing the same zinc ion content, zinc hyaluronate has a higher kinematic viscosity and light transmittance, while the osmotic pressure is lower.

[0157] Test Example 2.1 Patch Test

[0158] Experimental Materials

[0159] Sample 1: High molecular weight zinc hyaluronate sample (Example 9, 1270 kD);

[0160] Sample 2: Low molecular weight zinc hyaluronate sample (Example 10, 45 kD)

[0161] Experimental Procedure:

[0162] (1) Preparation of Samples

[0163] Purified water was used as a control. Sample 1 and Sample 2 were each prepared into concentrations of 1.0% and 0.5% with purified water.

[0164] (2) Skin Patch Test

[0165] Tear open the package of the patch tester, measure 0.025 mL of each prepared sample and add it to the chamber. Apply the patch tester to the flexor side of the forearm of the subject, and gently press it with the palm to make it evenly adhere to the skin for 24 h. There were 30 subjects.

[0166] 3. Result Analysis

[0167] Observe and record the reaction results at 30 min, 24 h, and 48 h after removing the patch tester according to Table 6.

[0168] Table 6 Skin Reaction Grading Criteria for Skin Closed Patch Test

[0169]

[0170] Note: Refer to the "Technical Specifications for Cosmetics Safety" - 2015 Edition.

[0171] Table 7 Statistical Results of Patch Test (Total Number of Cases: 30)

[0172]

[0173] The results are shown in Table 7. All samples of high and low molecular weight zinc hyaluronate at concentrations of 1.0% and 0.5% for 30 subjects were negative reactions, and there were no potential adverse reactions to the human body.

[0174] Test Example 2.2 Moisturizing Effect

[0175] Experimental materials:

[0176] Emulsion containing 0.5% zinc hyaluronate I (Example 9); Emulsion containing 0.5% zinc hyaluronate II (Example 10); Blank emulsion (without zinc hyaluronate)

[0177] The other components in the emulsion formulation are as shown in Table 8 below:

[0178] Table 8

[0179]

[0180] Experimental procedure:

[0181] Using the half-face control method, divided into two groups, A and B, with 10 people in each group;

[0182] Group A: The left face was treated with the emulsion containing high molecular weight zinc hyaluronate (Example 9), and the right face was treated with the blank emulsion; Group B: The left face was treated with the emulsion containing low molecular weight zinc hyaluronate (Example 10), and the right face was treated with the blank emulsion; The water content of the stratum corneum in the malar area of the subjects' faces was measured before and after treatment.

[0183] Experimental results:

[0184] The results of Group A and Group B are respectively as Figure 1A 、 1B shown. The initial value before use was set to 100%. Compared with the blank group, both high molecular weight zinc hyaluronate and low molecular weight zinc hyaluronate could increase the water content of the stratum corneum (4% - 8%) within 4 weeks of use.

[0185] Test Example 2.3: Oil control effect

[0186] Experimental materials:

[0187] Two emulsions prepared in Test Example 2.2

[0188] Experimental procedure:

[0189] Using the half-face control method, divided into two groups, A and B, with 10 people in each group;

[0190] Group A: The left face was treated with the emulsion containing high molecular weight zinc hyaluronate (Example 9), and the right face was treated with the blank emulsion; Group B: The left face was treated with the emulsion containing low molecular weight zinc hyaluronate (Example 10), and the right face was treated with the blank emulsion; The sebum secretion amount on the forehead, the porphyrin content in the T-zone, and the texture area in the malar area of the subjects' faces were measured before and after treatment.

[0191] Experimental results:

[0192] 1. Influence of the test samples on skin oil content

[0193] The results of Group A and Group B are shown respectively as Figure 2A , 2B shown below. The results are expressed as the difference from the skin oil content before use. Figure 2A In , compared with the blank group, the skin oil content in the high molecular weight zinc hyaluronate group was slightly lower, and the average oil content decreased by 5.70 μg / cm 2 at the 4th week; while Figure 2B in , the effect of reducing oil in the low molecular weight zinc hyaluronate group was more significant. Especially after 1 week of use, the average skin oil content decreased by 13.11 μg / cm 2 compared with the blank group.

[0194] 2. Effect of the test sample on the porphyrin content in the T-zone

[0195] Porphyrin is a metabolite of microorganisms. There is a certain correlation between the skin porphyrin content and the oil content. In areas with higher oil content, the microbial metabolic activity is usually more vigorous, and the amount of porphyrin produced is more. The results of Group A and Group B are shown respectively as Figure 3A , 3B shown below, and the initial value before use was set to 100%. Figure 3A In , compared with the blank group, zinc hyaluronate I could reduce the porphyrin content in the T-zone to a certain extent (reduce by 4% - 6%) only after 2 weeks of use; Figure 3B in , compared with the blank group, zinc hyaluronate II maintained a relatively low porphyrin content in the T-zone (reduce by 7% - 14%) throughout the 4 weeks of use.

[0196] Test Example 2.4: Inhibiting harmful skin bacteria

[0197] Experimental materials:

[0198] Zinc hyaluronate sample (Example 9, 1270 kD)

[0199] Experimental procedure:

[0200] 1) Appropriately dilute the test bacterial suspension with PBS solution, and the required concentration is: take 0.1 mL and drop it into 5.0 mL of the control sample solution (PBS phosphate buffer solution), and the recovered bacteria count is 1×10 4 ~9×10 4 cfu / mL.

[0201] 2) Dilute the test sample with sterile standard hard water to the specified concentration.

[0202] 3) Pipette 5.0 mL of the test sample stock solution or its dilution into a sterilized test tube and keep it at a constant temperature of 20°C for 5 minutes.

[0203] 4) Pipette 0.1 mL of the test bacterial solution into the test tube containing 5.0 mL of the sample, mix quickly, and start timing immediately.

[0204] 5) After the action reaches the set time, take 0.5 mL of the test bacteria and sample mixture, add it to a test tube containing 4.5 mL of sterilized PBS, and mix well.

[0205] 6) After standing for 10 min, pipette 1 mL of the sample solution (or, after appropriate dilution, take the dilution solutions of 2 - 3 dilution factors), place it in a sterilized petri dish, and inoculate two sterilized petri dishes for each sample solution or dilution factor. Pour 15 mL of nutrient agar medium (for bacteria) or Sabouraud agar medium (for Candida albicans) cooled to 40°C - 45°C, rotate the petri dish to make it fully uniform. After the agar solidifies, invert the petri dish and incubate at (35 ± 2)°C for 48 h (for bacteria) or 72 h (for Candida albicans), and then perform viable colony counting.

[0206] 7) Use PBS instead of the test sample and operate according to the above steps simultaneously as the control sample.

[0207] 8) Calculate the antibacterial rate

[0208]

[0209] Table 9

[0210]

[0211]

[0212] The results are shown in Table 9: After treatment with 0.5% zinc hyaluronate solution for 8 h, the inhibition rates against Staphylococcus epidermidis, Malassezia furfur, and Propionibacterium acnes are 50.03%, 72.06%, and 62.10% respectively. Therefore, zinc hyaluronate has an inhibitory effect on harmful bacteria on the skin surface, can prevent infections of skin wounds caused by skin boosters, microneedles, etc.; it can also be used in products for anti-dandruff (caused by Malassezia furfur) and anti-acne (caused by Propionibacterium acnes).

[0213] Test Example 2.5 Antioxidant Efficacy

[0214] (1) Hydroxyl Radical Scavenging Effect

[0215] 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 Co., Ltd., 1230 kD), 0.5% zinc acetate (dihydrate) solution;

[0216] Experimental method:

[0217] The salicylic acid method was used to determine the scavenging ability of zinc hyaluronate samples against hydroxyl radicals. A certain amount of H2O2 was mixed with FeSO4 to undergo the Fenton reaction to generate hydroxyl radicals. Salicylic acid was added to the reaction system, which could capture hydroxyl radicals and produce 3-hydroxybenzoic acid and 5-hydroxybenzoic acid. Both products had strong absorption at 510 nm. The reaction equation was as follows:

[0218] H2O2 + Fe 2+ →·OH + OH - + Fe 3+

[0219] The addition of the sample would compete with salicylic acid for the reaction with ·OH, thereby reducing the production amounts of 3-hydroxybenzoic acid and 5-hydroxybenzoic acid. The fixed reaction time method was adopted to measure the absorbance value of the reaction solution at 510 nm and compare it with the blank solution to determine the scavenging effect of the analyte on hydroxyl radicals.

[0220] The scavenging rate was calculated according to the following formula:

[0221] S / % = A0 - (A x - A x0 ) / A0 × 100

[0222] In the formula, S was the scavenging rate, A0 was the absorbance value of the blank control, A x was the absorbance value when the sample was added, and A x0 was the absorbance value without the color developing agent.

[0223] Experimental results:

[0224] Figure 4 The results showed that HA-Zn had an obvious effect on scavenging hydroxyl radicals, and the scavenging rate of low-molecular-weight zinc hyaluronate on hydroxyl radicals was higher, up to 85%; at the same concentration, HA-Na and zinc acetate dihydrate alone also had a certain ability to scavenge hydroxyl radicals, but the scavenging rate was significantly lower than that of HA-Zn. Therefore, zinc hyaluronate had a significant effect on scavenging hydroxyl radicals, reflecting that zinc hyaluronate had good antioxidant effects and was of great significance for maintaining the normal physiological activities of the body and anti-aging.

[0225] (2) Scavenging effect on superoxide anion

[0226] Experimental materials:

[0227] 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 Co., Ltd., 1230 kD), 0.5% zinc acetate (dihydrate) solution

[0228] Experimental method:

[0229] The pyrogallol autoxidation method was used for determination. Under weakly alkaline conditions, pyrogallol can undergo autoxidation reaction to generate superoxide anions, and at the same time generate a colored intermediate product, which has a characteristic absorption peak at 320 nm. In the initial stage, the amount of the intermediate product is linearly related to time. Since the autoxidation rate depends on the concentration of superoxide anions, when a superoxide anion scavenger is added, it can quickly react with superoxide anions and inhibit the autoxidation reaction, thereby preventing the accumulation of the intermediate product and weakening the light absorption of the solution at 320 nm. Therefore, the scavenging effect of the scavenger on superoxide anions can be evaluated by measuring the A320 value.

[0230] The clearance rate was calculated according to the following formula:

[0231] Superoxide anion radical scavenging rate (%) = (A0 - A x ) / A0 × 100%

[0232] It can be seen from Figure 5 the results that HA-Zn has a significant effect on scavenging superoxide anion radicals, among which the high-molecular-weight zinc hyaluronate has a higher scavenging ability, and the clearance rate can reach more than 70%. Although zinc acetate has a certain ability to scavenge superoxide anion radicals, 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 on scavenging superoxide anion radicals, which can reflect that zinc hyaluronate has a good antioxidant effect.

[0233] Test Example 2.6 Reduction of scar formation

[0234] Experimental materials: Zinc hyaluronate samples (Example 9, 1270 kD)

[0235] Experimental method:

[0236] According to the amino acid differences of the three peptide chains that make up procollagen, collagen can be divided into four types: I, II, III, and IV. Normal skin collagen is mainly type I and type III, and the content ratio of the two is about 3.5:1. During scar formation, both the absolute value and the relative proportion of type II collagen fibers show a decreasing trend, while type I collagen shows an increasing trend. Type I collagen is thick fiber and is the material basis for scar tissue fibrosis; type II collagen is thin fiber and is the main component of reticular fibers. With the formation of scars, type II fibers are gradually replaced by thick type I fibers, which destroys the reticular structure of normal skin, and the biological characteristics of normal skin change accordingly. The I / III collagen ratio drops significantly, the collagen fibers are disorderly arranged, and the fibers become thick, thus showing the characteristic appearance of scar formation.

[0237] Fibroblasts were treated with TGF-β1 to establish an in vitro scar model. The scar model was treated with a test substance working solution of zinc hyaluronate at 0.05 mg / ml, and images were collected using a fluorescence microscope within 48 h. The magnification of the images was 400 times (eyepiece 10×, objective 40×). The scar repair efficacy was evaluated by observing the changes in the contents of Collagen I and Collagen III in fibroblasts. (This test was conducted by Guangdong Boxi Biotechnology Co., Ltd.)

[0238] BC was the positive control, and NC was the negative (blank) control. It was known from Figure 6A and Figure 6B that compared with the NC group, after the sample zinc hyaluronate was applied at a concentration of 0.05 mg / mL for 24 h, the content of Collagen I decreased extremely significantly (P < 0.05); the content of Collagen III decreased, but there was no significant difference.

[0239] In summary, in the in vitro culture system, the sample zinc hyaluronate at a concentration of 0.05 mg / mL could significantly inhibit the synthesis of Collagen I, thereby affecting the protein expression ratio of Collagen I and Collagen III and inhibiting scar formation caused by the overexpression of Collagen I.

Claims

1. A method for preparing zinc hyaluronate, characterized in that, It includes the following steps: Adding sodium hyaluronate into an acidic aqueous organic medium containing a zinc salt for a single replacement 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; wherein the mass ratio of the addition amount of sodium hyaluronate to the zinc salt is 1:1 - 1:3, and the zinc ion concentration in the acidic aqueous organic medium containing the zinc salt is 1% - 3% w / v; wherein the concentration of the organic solvent in the acidic aqueous organic medium containing the zinc salt is 55% - 95% v / v, the pH of the acidic aqueous organic medium containing the zinc salt is 5.0 - 6.9, and the organic solvent is ethanol.

2. The preparation method according to claim 1, characterized in that, The concentration of the organic solvent in the acidic aqueous organic medium containing the zinc salt is 55% - 70% v / v.

3. The preparation method according to claim 1, characterized in that, The molecular weight of the sodium hyaluronate is 1 kDa - 3000 kDa.

4. The preparation method according to any one of claims 1-3, characterized in that, The replacement time is 1 - 24 h.

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