Preparation method and application of zinc hyaluronate

Abstract

The invention provides a preparation method of zinc hyaluronate, which comprises the following steps: adding sodium hyaluronate into an acidic water-containing organic medium containing zinc salt, and carrying out primary replacement to obtain a zinc hyaluronate precipitate; washing the precipitate with a washing liquid; and dehydrating the washed precipitate with a dehydration liquid, and drying to obtain the zinc hyaluronate powder. On the basis of a solid replacement method, the concentration of zinc salt in the replacement liquid, the volume of the replacement liquid, the replacement time and the number of replacement times are optimized, the problems that in an existing zinc hyaluronate production process, the number of replacement times is large, consumed time is long, molecular weight consumption is large, and the utilization rate of zinc salt is low are solved, the production period is greatly shortened, and the replacement efficiency is improved; the contact time of the raw material and the acid displacement liquid is reduced, and the great reduction of the molecular weight of the product is avoided. The invention provides a method for rapidly and efficiently producing zinc hyaluronate, which is suitable for large-scale production of zinc hyaluronate.

Description

[0001] This case is a divisional application of a case with an application date of April 20, 2022, an invention name of "A preparation method and application of zinc hyaluronate", and an application number of 202210424292.6. Technical Field

[0002] The invention relates to a preparation method and application of zinc hyaluronate, and belongs to the technical field of hyaluronates. Background Art

[0003] Hyaluronic acid (HA) is a polysaccharide composed of glucuronic acid and acetylglucosamine disaccharide units. It has physiological functions such as moisturizing, nutrition, repairing and preventing damage, and is widely used in the field of cosmetics. Studies have shown that in aqueous solutions with a pH of 6.0 to 6.5, Zn 2+ It can combine with the oxygen-containing donor groups in HA to form zinc hyaluronate (Zn-HA) complex.

[0004] Typically, zinc hyaluronate is obtained by exchanging sodium ions in sodium hyaluronate with zinc ions in zinc salts.

[0005] A Hungarian patent reports a process for preparing powdered Zn-HA: sodium hyaluronate is dissolved in water, zinc salt is added, precipitated with an organic solvent, and then dehydrated and dried with the organic solvent. Currently, the ion exchange resin method is commonly used in China: sodium hyaluronate is dissolved and applied to an ion exchange column to convert the sodium hyaluronate into zinc hyaluronate, which is then precipitated with ethanol and dehydrated. Both methods share the common principle of dissolving the sodium hyaluronate first and then performing ion exchange in the solution. The former method is simpler, but the product has a higher sodium ion content; the latter method requires more equipment and is more complex to operate, but the product has a higher zinc ion content.

[0006] Patent CN100355790C discloses a method for preparing zinc hyaluronate, which provides a method for preparing powdered zinc hyaluronate, wherein Na-HA solid is directly ion-exchanged with zinc salt in an alcohol solution to generate zinc hyaluronate.

[0007] Patent CN111647100A discloses a method for preparing high-molecular-weight zinc hyaluronate. By exploring various preparation conditions, including the pH of the replacement solution, the number of replacement cycles, the pH of the washes, the number of washes, the pH of the dehydration solution, and the number of dehydrations, the optimal preparation conditions were determined. The resulting zinc hyaluronate has a molecular weight exceeding 1,000,000 Da, a transmittance exceeding 99.5%, and a zinc content exceeding 7.0%.

[0008] The above processes all use multiple replacements, which have the problems of long cycle, large amount of organic solvent used, low zinc salt utilization rate, and large reduction in product molecular weight.

[0009] Zinc hyaluronate is the zinc salt of hyaluronic acid. In addition to its moisturizing properties, it also offers benefits such as anti-inflammatory, barrier repair, skin damage repair, skin texture improvement, and melanin reduction and whitening, making it suitable for cosmetics. Furthermore, zinc hyaluronate has physiological functions such as lubrication, antimicrobial properties, prevention and treatment of peptic ulcers, and promotion of wound repair, earning it widespread use in the medical field.

[0010] Patent CN102834417A discloses a method for producing a metal hyaluronic acid salt, a method for producing cosmetics containing a metal hyaluronic acid salt, and zinc hyaluronate and its production method. The skin is treated with a tape stripping method to disrupt the skin's barrier function. Then, an essence containing 0.1% zinc hyaluronate is applied. Experimental results show that zinc hyaluronate has an excellent effect on restoring transepidermal water evaporation and effectively repairing the skin barrier. It also significantly improves skin redness.

[0011] Patent CN112691049A discloses a shampoo composition containing zinc hyaluronate. Zinc hyaluronate is used to significantly inhibit Malassezia fungus on the scalp, providing anti-dandruff and anti-itching benefits. It can also be used in combination with other ingredients to provide oil control and soothing effects.

[0012] Patent CN102961396A discloses the use of hyaluronate in the preparation of drugs for treating skin diseases, its drug combination, and its preparation method. It provides a pharmaceutical composition (cream) containing zinc hyaluronate and / or sodium hyaluronate, in which the hyaluronate metal salt, especially the preparation with zinc hyaluronate as the main active ingredient, is effective in treating skin barrier dysfunction, and can regulate the physiological function of the skin and repair the skin barrier function. Summary of the Invention

[0013] To address long-standing problems in the production of zinc hyaluronate, the present invention optimizes the zinc salt concentration, displacement fluid volume, displacement time, and number of displacements in the displacement fluid based on a solid displacement method, thereby greatly shortening the production cycle, improving the displacement efficiency, reducing the contact time between the raw materials and the acidic displacement fluid, and avoiding a significant reduction in the product molecular weight.

[0014] In view of the problems existing in the current preparation methods of zinc hyaluronate, the present invention provides a method for quickly and efficiently producing zinc hyaluronate, which is suitable for large-scale production of zinc hyaluronate.

[0015] Specifically, the present invention adopts the following technical solutions:

[0016] 1. A method for preparing zinc hyaluronate, comprising the following steps:

[0017] Sodium hyaluronate is added to an acidic aqueous organic medium containing zinc salt to perform a displacement to obtain zinc hyaluronate precipitate;

[0018] washing the precipitate with a washing solution;

[0019] The washed precipitate is dehydrated with a dehydrating liquid and dried to obtain zinc hyaluronate powder.

[0020] 2. According to the preparation method of claim 1, the mass ratio of the added amount of sodium hyaluronate to the zinc salt is 1:0.5-1:3.5, preferably 1:1-1:3.

[0021] 3. According to the preparation method of item 1 or 2, the organic solvent concentration in the acidic aqueous organic medium containing 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 zinc salt is 5.0 to 6.9.

[0022] 4. The preparation method according to any one of items 1 to 3, wherein the molecular weight of the sodium hyaluronate is 1 kDa-3000 kDa.

[0023] 5. The preparation method according to any one of items 1 to 4, wherein the replacement time is 1 to 24 hours.

[0024] 6. According to the preparation method described in any one of items 1-5, the concentration of the organic solvent in the washing liquid is 70% to 85% (v / v), the pH of the washing liquid is 5.0 to 6.9, the number of washings is 2 to 6 times, the concentration of the organic solvent in the dehydration liquid is greater than or equal to 90% (v / v), and the number of dehydrations is more than 2 times.

[0025] 7. The low molecular weight zinc hyaluronate prepared by the method according to any one of items 1 to 6, wherein the molecular weight of the low molecular weight zinc hyaluronate is preferably less than or equal to 1000 kDa.

[0026] 8. High molecular weight zinc hyaluronate prepared by the method according to any one of items 1 to 6, preferably, the molecular weight of the high molecular weight zinc hyaluronate is greater than 1000 kDa.

[0027] 9. Use of zinc hyaluronate prepared by the method according to any one of items 1 to 8 in skin moisturizing, skin oil control, inhibition of harmful skin bacteria, anti-oxidation, scar repair, scar inhibition, and prevention of skin wound infection.

[0028] Preferably, the use of inhibiting harmful skin bacteria includes use in removing acne and dandruff;

[0029] Preferably, the use in preventing skin wound infection includes preventing skin wound infection caused by hyaluronic acid injection and microneedle injection.

[0030] 10. Use of zinc hyaluronate prepared by the method according to any one of items 1 to 8 in skin care products, including scalp care.

[0031] 11. Use of zinc hyaluronate in reducing skin oiliness, wherein the zinc hyaluronate is preferably low molecular weight zinc hyaluronate.

[0032] More preferably, the molecular weight of the low molecular weight zinc hyaluronate is 2 kDa-1000 kDa, preferably 5 kDa-500 kDa.

[0033] 12. Use of zinc hyaluronate for reducing the porphyrin content in skin oils, wherein the zinc hyaluronate is preferably low molecular weight zinc hyaluronate.

[0034] More preferably, the molecular weight of the low molecular weight zinc hyaluronate is 2 kDa-1000 kDa, preferably 5 kDa-500 kDa.

[0035] 13. Use of zinc hyaluronate for scavenging hydroxyl radicals on the skin surface, wherein the zinc hyaluronate is preferably low molecular weight zinc hyaluronate.

[0036] More preferably, the molecular weight of the low molecular weight zinc hyaluronate is 2 kDa-1000 kDa, preferably 5 kDa-500 kDa.

[0037] 14. Use of zinc hyaluronate for scavenging superoxide anion free radicals on the skin surface, wherein the zinc hyaluronate is preferably high molecular weight zinc hyaluronate.

[0038] More preferably, the molecular weight of the high molecular weight zinc hyaluronate is 1000 kDa to 2500 kDa, preferably 1100 kDa to 2000 kDa.

[0039] Effects of the Invention

[0040] 1. Process Improvement: Based on the solid displacement method, the present invention optimizes the zinc salt concentration, displacement fluid volume, displacement time, and number of displacements in the displacement fluid, significantly shortening the production cycle, improving displacement efficiency, reducing the contact time between the raw material and the acidic displacement fluid, and avoiding a significant decrease in the product molecular weight. The present invention provides a method for rapidly and efficiently producing zinc hyaluronate, suitable for large-scale production of zinc hyaluronate.

[0041] 2. Skin care efficacy: The zinc hyaluronate prepared by the present invention has the effects of moisturizing, inhibiting harmful bacteria on the skin, repairing damage, controlling oil, resisting oxidation, removing fine lines, etc., and can be used for skin care. Zinc hyaluronate is weakly acidic, which can ensure a weakly acidic environment for the skin (including the scalp), while inhibiting the growth of some pathogenic microorganisms, protecting the skin from the invasion of various fungi, and repairing inflammation and damage to the skin. In addition, zinc hyaluronate can keep the skin moist, protect and strengthen the skin's natural protective barrier, has a certain oil control effect, regulates the secretion of oil by the sebaceous glands, and maintains the water-oil balance. Therefore, zinc hyaluronate plays an important role in maintaining a healthy skin (including the scalp) ecological environment.

[0042] 3. The efficacy of zinc hyaluronate is increasingly being discovered, but its research and application in the pharmaceutical field are limited. There is no systematic and comprehensive study of the effects of zinc hyaluronate raw materials. Therefore, the present invention conducts a series of studies on the efficacy of zinc hyaluronate with different molecular weights, which has far-reaching significance for the application of zinc hyaluronate and the development of the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1A Effects of zinc hyaluronate I on the water content of the stratum corneum;

[0044] Figure 1B Effects of zinc hyaluronate II on stratum corneum water content;

[0045] Figure 2A Effects of zinc hyaluronate I on skin oil content;

[0046] Figure 2B Effects of zinc hyaluronate II on skin oil content;

[0047] Figure 3A Effects of Hyaluronate Zinc I on T-zone Porphyrin Content;

[0048] Figure 3B Effects of zinc hyaluronate II on porphyrin content in the T zone;

[0049] Figure 4 The scavenging effect of zinc hyaluronate on hydroxyl free radicals;

[0050] Figure 5 The scavenging effect of zinc hyaluronate on superoxide anion free radicals;

[0051] Figure 6A Changes in collagen I content;

[0052] Figure 6B Changes in collagen III content. DETAILED DESCRIPTION

[0053] The present invention is further described below with reference to the examples. It should be understood that the examples are only used to further illustrate and explain the present invention and are not intended to limit the present invention.

[0054] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and not restrictive. The present invention is further described below with reference to specific examples, but is not intended to limit the scope of the invention.

[0055] The method for rapidly and efficiently producing zinc hyaluronate according to the present invention comprises the following steps:

[0056] preparing an acidic aqueous organic medium containing zinc salt;

[0057] Soaking the sodium hyaluronate solid in an acidic aqueous organic medium containing zinc salt, stirring and replacing, and controlling the replacement time to achieve the required zinc ion content;

[0058] After the zinc ion content reaches the requirement, an acidic aqueous organic medium is added to the residue after removing the supernatant to wash away excess ions;

[0059] After washing, the product is dehydrated and then vacuum dried to obtain zinc hyaluronate.

[0060] 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 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.

[0061] In certain preferred embodiments, the zinc salt is zinc chloride, zinc acetate, zinc sulfate, zinc nitrate or zinc lactate.

[0062] In the present invention, the organic medium is an organic medium that has good compatibility with water but in which sodium hyaluronate or zinc hyaluronate is insoluble or slightly soluble, such as an alcohol organic solvent, a ketone organic solvent, an amide solvent or acetonitrile, preferably an alcohol organic solvent or a ketone organic solvent.

[0063] Exemplary alcohol organic solvents include, but are not limited to, methanol, ethanol, isopropanol, 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.

[0064] In certain preferred embodiments of the present invention, the alcohol organic solvent is methanol and ethanol.

[0065] Exemplary ketone solvents include, but are not limited to, methyl ethyl ketone, methyl isobutyl ketone, 1-methyl-2-pyrrolidone, cyclohexanone, acetone, and the like.

[0066] In certain preferred embodiments of the present invention, the ketone organic solvent is acetone.

[0067] Exemplary amide solvents include, but are not limited to, N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, or formamide.

[0068] In the present invention, the acidic aqueous organic medium refers to an aqueous organic medium prepared by dissolving any of the organic media described above in water to form a certain proportion, and the pH value of the aqueous organic medium is adjusted to make it acidic. The substance for adjusting the pH value of the aqueous organic medium can be hydrochloric acid, glacial acetic acid, sulfuric acid, phosphoric acid, etc.

[0069] In certain preferred embodiments of the present invention, the pH value of the acidic aqueous organic medium is in the range of 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, or 6.9.

[0070] In an acidic environment, the sodium ion dissociation of solid sodium hyaluronate is high, making ion replacement more likely. However, if the pH is too low, the sodium hyaluronate will degrade, affecting the product's molecular weight. Furthermore, the longer the product is in contact with acidic aqueous media, the more the molecular weight decreases. Therefore, controlling the appropriate pH and production cycle is crucial.

[0071] In certain preferred embodiments of the present invention, the mass concentration of the organic medium is 55 to 95 wt %, preferably 55 to 70 wt %.

[0072] Within this concentration range, sodium hyaluronate is insoluble and in solid form. The lower the concentration of the aqueous medium, the higher the degree of dispersion of sodium hyaluronate therein, but the sedimentation is slow, the loss is high, and the cycle is long; conversely, the dispersion is low, the loss is small, and the sedimentation is fast.

[0073] The concentration of zinc ions in the acidic aqueous organic medium containing zinc salt is 1 to 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%, or 3 wt%.

[0074] The concentration of zinc ions is related to the concentration of the organic medium in the acidic aqueous organic medium. The higher the organic medium concentration, the lower the zinc ion solubility. Therefore, under the appropriate concentration of the aqueous organic medium, the zinc ion concentration is preferably 1 to 3 wt%. This concentration can ensure that the zinc ion concentration reaches a certain level, can effectively promote the displacement reaction, and can ensure that the zinc salt is completely dissolved in the acidic aqueous organic medium.

[0075] 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 values. In a preferred embodiment of the present invention, the molecular weight of the sodium hyaluronate is 1 kDa to 3000 kDa, preferably 10 kDa to 2500 kDa, and the uronic acid content is 40 to 50 wt%, preferably 45 to 50 wt%.

[0076] Because degradation may occur during the preparation process, the molecular weight of the selected sodium hyaluronate should be higher than the required molecular weight of zinc hyaluronate.

[0077] The mass ratio of sodium hyaluronate to zinc salt 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, preferably 1:1-1:3.

[0078] The stirring and replacement time is 1-24 hours, preferably 5-16 hours.

[0079] Sodium hyaluronate is subjected to a displacement reaction in an acidic aqueous organic medium containing zinc salt. The higher the zinc ion concentration in the acidic aqueous organic medium, the larger the total amount of zinc salt used, and the longer the displacement time, the higher the zinc ion content in the product, that is, the higher the displacement rate. According to the content of zinc salt in the acidic aqueous medium, the volume of the displacement fluid is controlled by controlling the total amount of zinc salt used. When the ratio of the mass of sodium hyaluronate to the mass of zinc salt is 1: (0.5-3.5), the displacement efficiency is high. The longer the stirring time of the displacement, the higher the displacement efficiency. However, the longer the time, the more the product degrades and the more the molecular weight decreases. Therefore, the displacement time is controlled to be 1 to 24 hours, and more preferably, the displacement time is 5 to 16 hours.

[0080] Furthermore, in the above preparation method, the pH of the acidic aqueous organic medium in 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.

[0081] Furthermore, after the zinc ions are replaced to meet the requirements, the product is washed to remove excess unbound ions and stabilize the bound ions. The washing is performed with an acidic aqueous organic medium, wherein the acidic aqueous organic medium is an organic medium with good compatibility with water but in which sodium hyaluronate or zinc hyaluronate is insoluble or slightly soluble, preferably an alcoholic organic solvent or a ketone organic solvent, commonly used being ethanol, methanol, acetone, etc.

[0082] In the acidic aqueous organic medium, the concentration of the organic medium is 70-85 wt % and the pH is 5.0-6.9.

[0083] The standard for washing is to remove all excess ions.

[0084] In order to improve the washing effect and reduce the generation of waste water, washing can be carried out by multiple washing, stirring and soaking, etc.

[0085] Furthermore, after washing is completed, the product is dehydrated using a neutral aqueous organic medium, the concentration of which is greater than or equal to 90 wt %; the organic medium is an organic medium that has good compatibility 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, commonly used being ethanol, methanol, acetone, and the like.

[0086] After dehydration, the aqueous organic medium in the supernatant is removed and the mixture is dried to obtain solid zinc hyaluronate.

[0087] The drying method is vacuum drying, and the drying temperature can be adjusted according to the molecular weight requirements and drying loss requirements of the product, generally 20 to 75°C.

[0088] Furthermore, the zinc hyaluronate obtained by the above preparation method has a zinc ion content of 6.0-9.0 wt%, a replacement rate of 75%-100%, and other indicators: pH 5.5-7.5; loss on drying ≤15.0%, transmittance ≥99.0%, heavy metal ≤20 ppm, protein ≤0.1%, hyaluronate zinc content of more than 90 wt%, and hyaluronate zinc yield of more than 90%.

[0089] Furthermore, in the preparation process of zinc hyaluronate, in addition to the steps of stirring and replacing, washing, dehydrating, and drying, a step of degrading sodium hyaluronate may also be included. The degradation step is performed before stirring and replacing.

[0090] The degradation of sodium hyaluronate can be achieved by any method disclosed in the prior art, such as enzymatic hydrolysis, alkaline hydrolysis, etc.

[0091] 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 reduce the molecular weight of sodium hyaluronate to a desired molecular weight, and then the degraded sodium hyaluronate is stirred and replaced to prepare zinc hyaluronate.

[0092] The pH of degradation is different from the pH of stirring and replacement. The pH of degradation is less than 5, preferably greater than 1 and less than 5.

[0093] After the degradation is completed, the acidic aqueous organic medium containing zinc salt is added to the sodium hyaluronate after adjusting the pH with sodium hydroxide, and then stirred for replacement.

[0094] In the present application, the preparation method of the zinc hyaluronate, based on the existing preparation method of zinc hyaluronate, has been further optimized through multiple experimental attempts, by adjusting the mass ratio of sodium hyaluronate and zinc salt replacement process to a suitable ratio during the preparation process, so that sodium hyaluronate and zinc salt can achieve good replacement effect by only one replacement, avoiding the need to repeatedly add zinc salt to carry out multiple replacements in the prior art, consuming manpower and material resources, the present application further optimizes the concentration of the solvent used in the preparation process and the molecular weight of sodium hyaluronate, strengthens process parameters, and more ensures the quality of the zinc hyaluronate prepared. The preparation method of zinc hyaluronate of the present application simplifies preparation step, saves resources, and the zinc hyaluronate effect prepared is good, and transmittance and yield all have obvious advantages.

[0095] By regulating the degradation steps and selecting the molecular weight of sodium hyaluronate, zinc hyaluronate with different molecular weight ranges within the range of 1kDa to 2000kDa can be obtained, such as 1kDa-5kDa, 5kDa-10kDa, 10kDa-200kDa, 200kDa-500kDa, 500kDa-1000kDa, and 1000kDa-2000kDa.

[0096] Furthermore, the zinc hyaluronate prepared by the present invention has the functions of moisturizing, inhibiting harmful bacteria on the skin, repairing damage, controlling oil, anti-oxidation, and removing fine lines, maintaining a healthy skin (including scalp) ecological environment, and can be used as a raw material in the cosmetics field.

[0097] Zinc hyaluronate is the zinc salt of hyaluronic acid. It combines the excellent properties of HA with unique physiological functions and efficacy, and different molecular weight zinc hyaluronates exhibit varying biological activities. Low-molecular-weight zinc hyaluronate, also known as hydrolyzed zinc hyaluronate, refers to zinc hyaluronate with a molecular weight below 1000 kDa. It is obtained by the exchange reaction of hydrolyzed sodium hyaluronate with zinc ions. Experimental studies have shown that zinc hyaluronate has a significant inhibitory effect on various skin surface bacteria (such as Staphylococcus epidermidis, Malassezia, and Propionibacterium acnes), with high-molecular-weight zinc hyaluronate being more effective than hydrolyzed zinc hyaluronate. Zinc hyaluronate also has the effect of reducing epidermal sebum. Porphyrins are microbial metabolites, and skin porphyrin levels correlate with lipid levels. Areas with higher lipid levels typically experience more active microbial metabolic activity, resulting in higher porphyrin production. Zinc hyaluronate can reduce porphyrin levels in the T-zone to a certain extent, with low-molecular-weight zinc hyaluronate being more effective. Low-molecular-weight zinc hyaluronate can also reduce fine lines, improve skin texture, and delay skin aging. Furthermore, 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 formation of scars caused by excessive expression of Collagen I.

[0098] Furthermore, the zinc hyaluronate can be used to prepare skin care products. The skin care products include moisturizing, antibacterial, anti-inflammatory, oil control, anti-aging, and repair.

[0099] Furthermore, the skin care products are toner, essence, gel, lotion, cream, facial mask, makeup, soap, facial cleanser, shampoo, conditioner and shower gel.

[0100] Furthermore, skin care products of various dosage forms containing zinc hyaluronate are all protected by the present invention.

[0101] The zinc hyaluronate prepared in the present application is used in skin moisturizing, skin oil control, inhibition of harmful skin bacteria, anti-oxidation, scar repair, scar inhibition, and prevention of skin wound infection.

[0102] Through extensive experimental research, the present application has found that skin porphyrin content is somewhat correlated with oil content. Regions with higher oil content typically experience more active microbial metabolic activity, resulting in higher porphyrin production. The zinc hyaluronate prepared herein can significantly maintain skin porphyrin content at a lower level, thereby effectively controlling skin oil content. In a preferred embodiment, the zinc hyaluronate has a molecular weight between 2kDa and 1000kDa, with a more optimal oil-control effect, preferably between 5kDa and 500kDa.

[0103] The zinc hyaluronate prepared in this application has a good effect in removing acne and dandruff. Further research in this application has found that the zinc hyaluronate has a high inhibition rate on Staphylococcus epidermidis, which can reach about 50%. At the same time, the inhibition rate on Malassezia furfur can reach more than 70%. Malassezia furfur mainly causes dandruff and dandruff problems. Therefore, the zinc hyaluronate of this application has a good use in removing dandruff. The zinc hyaluronate also has an inhibition rate of more than 60% on Propionibacterium acnes, which mainly causes acne problems. Therefore, the zinc hyaluronate of this application also has a good use in removing acne. Furthermore, this application can also prevent infection of skin wounds caused by hydrating needles, microneedles, etc.

[0104] The hyaluronic acid prepared in the present application has excellent anti-oxidation applications. The zinc hyaluronate has a significant effect on scavenging hydroxyl radicals, and the low-molecular-weight zinc hyaluronate has a higher hydroxyl radical scavenging rate, which can reach more than 80%. In a preferred embodiment, the molecular weight of the low-molecular-weight zinc hyaluronate is better between 2kDa and 1000kDa, and more preferably between 5kDa and 500kDa.

[0105] The present application further discovered that the zinc hyaluronate has a good use in scavenging superoxide anion free radicals from the skin surface. The zinc hyaluronate has a significant effect on scavenging superoxide anion free radicals, wherein high-molecular-weight zinc hyaluronate has a higher scavenging ability, with a scavenging rate of over 70%. In a preferred embodiment, the high-molecular-weight zinc hyaluronate has a molecular weight between 1000kDa and 2500kDa, which is more effective, and more preferably between 1100kDa and 2000kDa.

[0106] Beneficial effects

[0107] 1. In the present invention, the preparation of zinc hyaluronate can be achieved through only one replacement, and the zinc ion content in the prepared zinc hyaluronate is greater than 4wt%, the replacement rate is greater than 56%, the transmittance is greater than 88.0%, and the yield is greater than 89%.

[0108] 2. The preparation of zinc hyaluronate of the present invention is fast and efficient, saving a lot of production time and manpower and material costs. The obtained zinc hyaluronate has a high yield and is suitable for large-scale industrial production.

[0109] Example

[0110] The present invention is described in detail below using examples. However, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough understanding of the present invention and to enable the scope of the present invention to be fully conveyed to those skilled in the art. The numerical ranges cited in the present invention all include the data of the two endpoints of this numerical range, also include each specific numerical value in this numerical range, and this numerical value can be arbitrarily combined with the endpoints to form a new small range.

[0111] In the following examples and comparative examples, the sodium hyaluronate raw materials used were all from Bloomage Biotechnology Co., Ltd.

[0112] In the following examples, the uronic acid content was detected by sulfuric acid-carbazole colorimetry, the sodium content and zinc content were detected by atomic absorption spectrophotometry, and the molecular weight was detected by intrinsic viscosity method.

[0113] In the following examples, the replacement rate refers to the replacement efficiency of zinc ions in zinc hyaluronate, and the replacement rate is calculated as the ratio of the actual detected value of zinc ions in zinc hyaluronate to the theoretical value of complete replacement of zinc ions.

[0114] In the following examples, the yield is the ratio of the discharge amount to the input amount, and the calculation formula is:

[0115] Yield = Hyaluronate zinc output / Sodium hyaluronate input × 100%

[0116] In the following examples, the transmittance of a 0.5% aqueous solution was measured at 550 nm using a spectrophotometer.

[0117] In the following examples, unless otherwise specified, the concentrations are all expressed in mass percentage.

[0118] Example 1

[0119] Reagents: Ethanol, Bloomage Biotech Co., Ltd.

[0120] Glacial acetic acid, Sinopharm Chemical Reagent Co., Ltd.;

[0121] Sodium hyaluronate, Bloomage Biotech Co., Ltd.;

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

[0123] Zinc chloride, Sinopharm Chemical Reagent Co., Ltd.;

[0124] Zinc sulfate, Sinopharm Chemical Reagent Co., Ltd.

[0125] 170 L of a zinc acetate-ethanol solution containing 1.88 wt % zinc ions was prepared, wherein the ethanol concentration was about 55 wt %, and the pH was adjusted to 6.5 with glacial acetic acid.

[0126] Accurately weigh 30 kg of sodium hyaluronate (molecular weight 2500 kDa) solid, add it to the above replacement solution, start stirring to carry out replacement, and stir and replace for 8 hours.

[0127] The mixture was allowed to stand until the supernatant became clear, the supernatant was removed, and 800 L of a 70 wt% ethanol aqueous solution (pH 6.3) was added for washing. The mixture was stirred for 3 h, and then allowed to stand until the supernatant became clear, the supernatant was removed, and 800 L of a 70 wt% ethanol aqueous solution (pH 6.3) was added for washing in the same manner for a total of 4 washes.

[0128] After the last washing, the supernatant was removed and 800 L of 90 wt% ethanol solution was added for dehydration. Dehydration was performed twice.

[0129] Then the mixture was transferred to a three-in-one dryer for vacuum drying at a drying temperature of 45°C, a vacuum degree of 0.10 MPa, and a drying time of 18 h, and 28.33 kg of zinc hyaluronate was obtained.

[0130] Example 2-17

[0131] Hyaluronate zinc of Examples 2-17 was prepared according to the method of Example 1. The amounts of various substances and technical parameters in the method are shown in the table. Technical parameters not mentioned in the table are the same as those in Example 1.

[0132] Table 1 Contents of substances and technical parameters in each example

[0133]

[0134]

[0135] Test Example 1 Product Performance and Quality of Hyaluronate Zinc

[0136] The product performance and quality of the zinc hyaluronate prepared in Examples 1-18 were measured, and the various indicators are shown in Table 2. As can be seen from the table, by adjusting the reasonable ratios and concentrations of the components and appropriate parameter indicators, the present invention can achieve the preparation of zinc hyaluronate through a single replacement. The prepared zinc hyaluronate has a zinc ion content of greater than 4 wt%, a replacement rate of greater than 56%, a transmittance of greater than 88.0%, and a yield of greater than 89%, and the product quality indicators meet the requirements.

[0137]

[0138]

[0139] Test Example 2 Study on the Properties of Hyaluronate Zinc

[0140] Experimental Materials:

[0141] Zinc hyaluronate (Example 9, molecular weight 1270 kD), sodium hyaluronate (Huaxi Biotechnology Co., Ltd., molecular weight 1230 kD)

[0142] Experimental process:

[0143] Aqueous solutions of 0.5% zinc hyaluronate and 0.5% sodium hyaluronate (containing 0.13g zinc acetate dihydrate) were prepared, ensuring that the zinc ion content in both samples was 0.039%. The samples were named HA-Zn and HA-Na+Zn salt, respectively. The dynamic viscosity, transmittance, and osmotic pressure of the two solutions were tested.

[0144] Experimental results:

[0145] (1) Dynamic viscosity

[0146] The dynamic viscosity of the sample solution with a concentration of 0.5% was measured at 25°C. The results are shown in Table 3.

[0147] Table 3

[0148] Sample name HA-Zn HA-Na+Zn salt Dynamic viscosity (mPa·s) 310.0 260.0

[0149] (2) Light transmittance

[0150] The transmittance of the samples was measured at 550 nm using a spectrophotometer. The results are shown in Table 4.

[0151] Table 4

[0152] Sample name HA-Zn HA-Na+Zn salt Light transmittance (%) 94.62％ 81.26％

[0153] (3) Osmotic pressure

[0154] The osmotic pressure of the samples was measured with the osmotic pressure of water being 0. The results are shown in Table 5.

[0155] Table 5

[0156] Sample name HA-Zn HA-Na+Zn salt Osmolality (mOsm / kg) +10 +29

[0157] From the above results, it can be seen that compared with the sodium hyaluronate solution containing the same zinc ion content, the dynamic viscosity and transmittance of zinc hyaluronate are higher, while the osmotic pressure is lower.

[0158] Test Example 2.1 Patch test

[0159] Experimental Materials

[0160] Sample 1: high molecular weight zinc hyaluronate sample (Example 9, 1270 kD);

[0161] Sample 2: Low molecular weight zinc hyaluronate sample (Example 10, 45 kD)

[0162] Experimental process:

[0163] (1) Sample preparation

[0164] Purified water was used as a control, and sample 1 and sample 2 were prepared with purified water to concentrations of 1.0% and 0.5%, respectively.

[0165] (2) Skin patch test

[0166] Open the packaging of the patch tester, measure 0.025 mL of each prepared sample and add it to the chamber. Apply the patch tester to the curved side of the subject's forearm, gently press it with the palm of your hand to evenly apply it to the skin for 24 hours. 30 subjects were included.

[0167] 3. Results Analysis

[0168] Observe and record the reaction results according to Table 6 30 minutes, 24 hours and 48 hours after removing the spot tester.

[0169] Table 6 Skin reaction grading standards for skin occlusive patch test

[0170]

[0171] Note: Refer to the "Technical Specifications for Safety of Cosmetics" - 2015 edition.

[0172] Table 7 Patch test results statistics (total number of cases: 30)

[0173]

[0174] The results are shown in Table 7. All 30 subjects tested negative for 1.0% and 0.5% concentrations of high and low molecular weight zinc hyaluronate, indicating no potential adverse reactions to the human body.

[0175] Test Example 2.2 Moisturizing Effect

[0176] Experimental Materials:

[0177] Emulsion containing 0.5% hyaluronate zinc I (Example 9); Emulsion containing 0.5% hyaluronate zinc II (Example 10); Blank emulsion (not containing hyaluronate zinc)

[0178] Other ingredients in the emulsion formula are shown in Table 8 below:

[0179] Table 8

[0180]

[0181] Experimental process:

[0182] The half-face comparison method was used, and the subjects were divided into two groups, A and B, with 10 subjects in each group;

[0183] Group A: The left cheek was treated with an emulsion containing high molecular weight zinc hyaluronate (Example 9), and the right cheek was treated with a blank emulsion. Group B: The left cheek was treated with an emulsion containing low molecular weight zinc hyaluronate (Example 10), and the right cheek was treated with a blank emulsion. The water content of the stratum corneum in the apple muscle area of the subjects' faces was measured before and after treatment.

[0184] Experimental results:

[0185] The results of Group A and Group B are as follows: Figure 1A 、 1B As shown, the initial value before use was set as 100%. Compared with the blank group, high molecular weight zinc hyaluronate and low molecular weight zinc hyaluronate were able to increase the water content of the stratum corneum (4%-8%) within 4 weeks of use.

[0186] Test Example 2.3: Oil Control Effect

[0187] Experimental Materials:

[0188] Two emulsions prepared in Test Example 2.2

[0189] Experimental process:

[0190] The half-face comparison method was used, and the subjects were divided into two groups, A and B, with 10 subjects in each group;

[0191] Group A: The left cheek was treated with an emulsion containing high molecular weight zinc hyaluronate (Example 9), and the right cheek was treated with a blank emulsion. Group B: The left cheek was treated with an emulsion containing low molecular weight zinc hyaluronate (Example 10), and the right cheek was treated with a blank emulsion. The subjects' forehead oil secretion, T-zone porphyrin content, and facial texture area in the apple cheek region were measured before and after treatment.

[0192] Experimental results:

[0193] 1. Effect of test samples on skin oil content

[0194] The results of Group A and Group B are as follows: Figure 2A 、 2B The results are expressed as the difference between the skin oil content before use. Figure 2A Compared with the blank group, the skin oil content of the high molecular weight zinc hyaluronate group was slightly lower, and the average oil content decreased by 5.70 μg / cm at the fourth week. 2 ;and Figure 2BThe medium and low molecular weight zinc hyaluronate groups showed a more significant effect in reducing oil content. In particular, after one week of use, the average skin oil content was reduced by 13.11 μg / cm compared with the blank group. 2 .

[0195] 2. Effect of test samples on T-zone porphyrin content

[0196] Porphyrin is a metabolite of microorganisms. The content of porphyrin in skin is correlated with the oil content. In areas with higher oil content, microbial metabolic activity is usually more active, and the amount of porphyrin produced is higher. The results of Group A and Group B are as follows: Figure 3A 、 3B As shown, the initial value before use is set to 100%. Figure 3A In the study, compared with the blank group, zinc hyaluronate I could reduce the porphyrin content in the T zone to a certain extent (4%-6%) after 2 weeks of use; Figure 3B In the study, compared with the blank group, zinc hyaluronate II maintained a lower T-zone porphyrin content (reduced by 7%-14%) within 4 weeks of use.

[0197] Test Example 2.4: Inhibition of harmful skin bacteria

[0198] Experimental Materials:

[0199] Zinc hyaluronate sample (Example 9, 1270 kD)

[0200] Experimental process:

[0201] 1) Dilute the test bacterial suspension appropriately with PBS solution. The required concentration is: take 0.1 mL and drop it into 5.0 mL of control sample solution (PBS phosphate buffer solution). The number of recovered bacteria is 1×10 4 ~9×10 4 cfu / mL.

[0202] 2) Dilute the test sample with sterile standard hard water to the specified concentration.

[0203] 3) Pipette 5.0 mL of the test sample solution or its dilution into a sterile test tube and keep it at 20°C for 5 minutes.

[0204] 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 the time immediately.

[0205] 5) After the set time, take 0.5 mL of the test bacteria and sample mixture and add it to a test tube containing 4.5 mL of sterilized PBS and mix thoroughly.

[0206] 6) After 10 minutes, pipette 1 mL of the sample solution (or, after appropriate dilution, 2-3 dilutions thereof) into a sterile plate. Inoculate two sterile plates for each sample solution or dilution. Pour 15 mL of nutrient agar (for bacteria) or Sabouraud agar (for Candida albicans) cooled to 40-45°C, rotating the plate for thorough and uniform distribution. After the agar solidifies, flip the plate over. Incubate at (35±2)°C for 48 hours (for bacteria) or 72 hours (for Candida albicans), and count the viable colonies.

[0207] 7) Substitute PBS for the test sample and follow the above steps to serve as the control sample.

[0208] 8) Calculate the inhibition rate

[0209]

[0210] Table 9

[0211]

[0212] The results are shown in Table 9: After 8 hours of treatment with a 0.5% zinc hyaluronate solution, the inhibition rate against Staphylococcus epidermidis was 50.03%, against Malassezia furfur was 72.06%, and against Propionibacterium acnes was 62.10%. Therefore, zinc hyaluronate has an inhibitory effect on harmful bacteria on the skin surface, preventing infection caused by skin wounds such as dermatitis and microneedling. It can also be used in products for dandruff removal (caused by Malassezia furfur) and acne treatment (caused by Propionibacterium acnes).

[0213] Test Example 2.5 Antioxidant Efficacy

[0214] (1) Hydroxyl radical scavenging effect

[0215] Experimental materials: 0.5% hyaluronate zinc I (Example 9, 1270 kD) solution sample, 0.5% hyaluronate zinc II (Example 10, 45 kD) sample; 0.5% sodium hyaluronate solution sample (Bloomage Biosciences Co., Ltd., 1230 kD), 0.5% zinc acetate (dihydrate) solution;

[0216] Experimental methods:

[0217] The salicylic acid method was used to determine the hydroxyl radical scavenging ability of zinc hyaluronate samples. A certain amount of H₂O₂ was mixed with FeSO₄ to produce a Fenton reaction that generated hydroxyl radicals. Adding salicylic acid to the reaction system captured the hydroxyl radicals and produced 3-hydroxysalicylic acid and 5-hydroxysalicylic acid. Both products exhibited strong absorption at 510 nm. The reaction equations are as follows:

[0218] H2O2+Fe 2+ → OH+OH- +Fe 3+

[0219] The addition of the sample will compete with salicylic acid for the ·OH reaction, thereby reducing the production of 3-hydroxysalicylic acid and 5-hydroxysalicylic acid. Using the fixed reaction time method, the absorbance of the reaction solution at 510 nm is measured and compared with the blank solution to determine the scavenging effect of the analyte on hydroxyl radicals.

[0220] The clearance rate was calculated as follows:

[0221] S / %=A0-(A x -A x0 ) / A0×100

[0222] Where S is the clearance rate, A0 is the absorbance value of the blank control, and A x is the absorbance value when the sample is added, A x0 is the absorbance value without adding color developer.

[0223] Experimental results:

[0224] Figure 4 The results showed that HA-Zn had a significant effect on scavenging hydroxyl radicals, with low-molecular-weight zinc hyaluronate having a higher hydroxyl radical scavenging rate, reaching 85%. At the same concentration, HA-Na and zinc acetate dihydrate alone also had some hydroxyl radical scavenging ability, but at rates significantly lower than HA-Zn. Therefore, zinc hyaluronate has a significant hydroxyl radical scavenging effect, reflecting its excellent antioxidant properties and its important significance for maintaining normal physiological activities and anti-aging.

[0225] (2) Superoxide anion scavenging effect

[0226] Experimental Materials:

[0227] 0.5% hyaluronate zinc I (Example 9, 1270 kD) solution sample, 0.5% hyaluronate zinc II (Example 10, 45 kD) sample; 0.5% sodium hyaluronate solution sample (Bloomage Biosciences Co., Ltd., 1230 kD), 0.5% zinc acetate (dihydrate) solution

[0228] Experimental methods:

[0229] The determination is made using the pyrogallol autoxidation method. Under weakly alkaline conditions, pyrogallol undergoes autoxidation to generate superoxide anions and a colored intermediate with a characteristic absorption peak at 320 nm. In the initial phase, the amount of the intermediate is linearly proportional to time. Since the autoxidation rate depends on the concentration of superoxide anions, the addition of a superoxide anion scavenger rapidly reacts with the superoxide anions, inhibiting the autoxidation reaction and preventing the accumulation of the intermediate, resulting in a decrease in the solution's light absorption at 320 nm. Therefore, the A320 value can be used to evaluate the scavenging effect of a scavenger on superoxide anions.

[0230] The clearance rate is calculated as follows:

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

[0232] Depend on Figure 5 The results show that HA-Zn has a significant effect on scavenging superoxide anion radicals, with high-molecular-weight zinc hyaluronate exhibiting the highest scavenging capacity, exceeding 70%. While zinc acetate has some superoxide anion radical scavenging ability, it is significantly lower than that of HA-Zn, and HA-Na has almost no superoxide anion radical scavenging ability. Therefore, zinc hyaluronate has a significant superoxide anion radical scavenging effect, reflecting its excellent antioxidant properties.

[0233] Test Example 2.6 Reduction of scar formation

[0234] Experimental materials: Hyaluronate zinc sample (Example 9, 1270 kD)

[0235] Experimental methods:

[0236] Based on the amino acid differences in the three peptide chains that make up tropocollagen, collagen can be divided into four types: I, II, III, and IV. Normal skin collagen is primarily composed of types I and III, with a ratio of approximately 3.5:1. During scar formation, both the absolute value and relative proportion of type II collagen fibers decrease, while type I collagen increases. Type I collagen is a coarse fiber that forms the basis of scar tissue fibrosis; type II collagen is a fine fiber that is the primary component of the reticular fibers. As scarring forms, type II fibers are gradually replaced by coarse type I fibers, disrupting the reticular structure of normal skin and altering its biological properties. The type I / III collagen ratio decreases significantly, collagen fibers become disorganized, and fibers become coarse, resulting in the hallmark appearance of scarring.

[0237] An in vitro scar model was established by treating fibroblasts with TGF-β1. The scar model was treated with a 0.05 mg / ml working solution of zinc hyaluronate. Images were collected using a fluorescence microscope within 48 hours at a magnification of 400x (10x eyepiece, 40x objective). Scar repair efficacy was assessed by observing changes in Collagen I and Collagen III levels in fibroblasts. (This test was conducted by Guangdong Boxi Biotechnology Co., Ltd.)

[0238] BC is the positive control, NC is the negative (blank) control, Figure 6A and Figure 6B It can be seen that compared with the NC group, after the sample was treated with hyaluronate zinc at a concentration of 0.05 mg / mL for 24 hours, 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 hyaluronate zinc at a concentration of 0.05 mg / mL 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 excessive expression of Collagen I.

Claims

1. A method for preparing zinc hyaluronate, characterized in that: The following steps are involved: Sodium hyaluronate is added to an acidic aqueous organic medium containing zinc salt to perform a displacement 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 mass ratio of the added 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% to 3% w / v; The organic solvent concentration in the acidic aqueous organic medium containing zinc salt is 55% to 95% v / v, the pH of the acidic aqueous organic medium containing zinc salt is 5.0 to 6.9, and the organic solvent is an alcohol organic solvent, a ketone organic solvent, an amide solvent or acetonitrile.

2. The preparation method according to claim 1, characterized in that The organic solvent concentration in the acidic aqueous organic medium containing zinc salt is 55% to 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 to 3, characterized in that The replacement time is 1 to 24 hours.

5. The preparation method according to claim 1, characterized in that The washing liquid is an alcohol organic solvent, a ketone organic solvent, an amide solvent or acetonitrile.

6. The preparation method according to claim 5, characterized in that The concentration of the washing liquid is 70-85 wt%.

7. The preparation method according to claim 5, characterized in that The pH of the washing liquid is 5.0-6.

9.

8. The preparation method according to claim 1, characterized in that The dehydrating liquid is an alcohol organic solvent, a ketone organic solvent, an amide solvent or acetonitrile.

9. The preparation method according to claim 8, characterized in that The concentration of the dehydrating liquid is greater than or equal to 90 wt %.

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