WO2023125789A1

WO2023125789A1 - Calcium hyaluronate, and preparation method therefor and use thereof

Info

Publication number: WO2023125789A1
Application number: PCT/CN2022/143332
Authority: WO
Inventors: 冯宁; 宗文斌; 刘文君; 王秀娟; 吴佳婧; 毛华; 石艳丽; 郭学平
Original assignee: 华熙生物科技股份有限公司; 华熙生物科技(天津)有限公司
Priority date: 2021-12-31
Filing date: 2022-12-29
Publication date: 2023-07-06

Abstract

The present application provides a method for preparing calcium hyaluronate, the method comprising the following steps: adding sodium hyaluronate to an acidic displacement solution containing a calcium salt, and subjecting same to displacement to obtain a calcium hyaluronate precipitate; washing the precipitate with an acidic washing solution; and dehydrating the washed precipitate with a neutral dehydrating solution, and vacuum drying same to obtain a calcium hyaluronate powder, wherein the concentration of an organic medium in the displacement solution is 50-90 wt%, preferably 60-80 wt%; the concentration of the calcium salt is 0.5-4 wt%, preferably 1-3 wt%; the concentration of an organic medium in the washing solution is 50-90 wt%, preferably 60-80 wt%; and the concentration of an organic medium in the dehydrating solution is greater than or equal to 90 wt%. In the present application, the finished calcium hyaluronate product is obtained by means of high-efficiency displacement; and the problems of a high number of displacement times, a long duration, large consumption of calcium salt, low efficiency of calcium displacement, and turbidity of a calcium hyaluronate solution in the current calcium hyaluronate production process are solved.

Description

一种透明质酸钙及其制备方法和应用A kind of calcium hyaluronate and its preparation method and application

技术领域technical field

本申请属于生物医药领域，涉及一种透明质酸钙及其制备方法和应用。The application belongs to the field of biomedicine, and relates to calcium hyaluronate and its preparation method and application.

背景技术Background technique

透明质酸钙是透明质酸的钙盐，透明质酸是由N-乙酰氨基葡糖和D-葡糖醛酸双糖单位小红腹交替链接而成的高分子黏多糖，它广泛的存在与人体的各组织中，是皮肤、玻璃体、关节滑液和软组织的重要成分，具有独特的理化性质和生物学功能。其独特的黏弹性、保水性、渗透性。生物相容性及非免疫原性使其广泛的应用于眼科、骨科、化妆品和保健食品等多个领域。透明质酸钙在具有透明质酸保湿性，粘弹性外，还具有较好的水溶性，便于人体吸收，可作为保健产品或营养剂来为人体补充钙质，对关节炎起到一定预防作用，可增强人体免疫力及防止骨质疏松等功能，有具有修复组织损伤及保持关节润滑度可广泛应用于食品、保健品、医药、化妆品护肤等行业。Calcium hyaluronate is the calcium salt of hyaluronic acid. Hyaluronic acid is a polymer mucopolysaccharide formed by alternating links of N-acetylglucosamine and D-glucuronic acid disaccharide units. It exists widely Among the various tissues of the human body, it is an important component of skin, vitreous body, joint synovial fluid and soft tissue, and has unique physical and chemical properties and biological functions. Its unique viscoelasticity, water retention and permeability. Biocompatibility and non-immunogenicity make it widely used in many fields such as ophthalmology, orthopedics, cosmetics and health food. Calcium hyaluronate not only has hyaluronic acid moisturizing properties and viscoelasticity, but also has good water solubility, which is easy to be absorbed by the human body. It can be used as a health care product or nutrient to supplement calcium for the human body, and has a certain preventive effect on arthritis. , can enhance human immunity and prevent osteoporosis and other functions. It can repair tissue damage and maintain joint lubrication. It can be widely used in food, health care products, medicine, cosmetics and skin care industries.

透明质酸钙现有制备工艺为两种：专利CN 1563108A一种制备低分子透明质酸钙的方法是将透明质酸钠酸降至合适的分子量，溶解后活性炭吸附后过滤，液体上柱后通过离子交换，与钙盐溶液水浴加热后浓缩，最后经沉淀，脱水，干燥后得到透明质酸钙粉末成品。本方法工艺较为复杂，收率低，周期长，使用设备多，不适合车间大规模生产。专利CN 1326883C一种透明质酸钙的制备方法中是通过透明质酸钠与含有钙盐的有机介质，通过置换反应，将透明质酸钠中的钠离子置换为钙离子，制备出透明质酸钙成品，本方法制备过程中，置换次数较多，置换效率低，有机介质使用多，周期较长，且制备成品中可能残留较多的游离钙离子，透明质酸与钙离子结合不牢固，导致透明质酸钙产品质量不稳定，溶液浑浊。There are two existing preparation processes for calcium hyaluronate: patent CN 1563108A, a method for preparing low-molecular-weight calcium hyaluronate is to reduce sodium hyaluronate acid to a suitable molecular weight, dissolve it, filter it with activated carbon after adsorption, and put the liquid on the column Through ion exchange, it is heated in a water bath with calcium salt solution, concentrated, and finally precipitated, dehydrated, and dried to obtain the finished product of calcium hyaluronate powder. The process of this method is relatively complicated, the yield is low, the cycle is long, and many equipments are used, so it is not suitable for large-scale production in workshops. In the patent CN 1326883C, a method for preparing calcium hyaluronate is to replace sodium ions in sodium hyaluronate with calcium ions through sodium hyaluronate and an organic medium containing calcium salts through a displacement reaction to prepare hyaluronic acid Calcium finished product, in the preparation process of this method, the replacement times are many, the replacement efficiency is low, the organic medium is used more, the cycle is longer, and there may be more free calcium ions left in the finished product, the combination of hyaluronic acid and calcium ions is not firm, The quality of the calcium hyaluronate product is unstable and the solution is cloudy.

发明内容Contents of the invention

针对现有技术存在的上述问题，本申请提供一种透明质酸钙及其制备方法和应用。For the above-mentioned problems existing in the prior art, the application provides a kind of calcium hyaluronate and its preparation method and application.

具体来说，本申请涉及如下方面：Specifically, this application involves the following aspects:

1.一种透明质酸钙的制备方法，其特征在于，包括以下步骤：1. a preparation method of calcium hyaluronate, is characterized in that, comprises the following steps:

将透明质酸钠加入到含有钙盐的酸性置换液中，进行置换，得到透明质酸钙沉淀；adding sodium hyaluronate to the acidic replacement solution containing calcium salts for replacement to obtain calcium hyaluronate precipitates;

用酸性洗涤液洗涤所述沉淀；Washing the precipitate with an acid washing solution;

将洗涤后的所述沉淀用中性脱水液进行脱水，真空干燥，得到透明质酸钙粉末；Dehydrating the washed precipitate with a neutral dehydrating solution and drying it in vacuum to obtain calcium hyaluronate powder;

其中所述置换液中有机介质浓度为50％～90wt％，优选为60％～80wt％，钙盐浓度为0.5％～4wt％，优选为1％～3wt％。Wherein the replacement liquid has an organic medium concentration of 50%-90wt%, preferably 60%-80wt%, and a calcium salt concentration of 0.5%-4wt%, preferably 1%-3wt%.

2.根据项1所述的制备方法，其特征在于，所述有机介质为与水相容性好、但透明质酸钠或透明质酸钙在其中不溶或微溶的有机介质，优选为醇类有机溶剂或酮类有机溶剂，更优选为乙醇、甲醇或丙酮。2. according to the preparation method described in item 1, it is characterized in that, described organic medium is the organic medium that has good compatibility with water, but sodium hyaluronate or calcium hyaluronate is insoluble or slightly soluble therein, is preferably alcohol Organic solvents or ketone organic solvents, more preferably ethanol, methanol or acetone.

3.根据项1所述的制备方法，其特征在于，透明质酸钠的分子量为3kDa～2200kDa。3. The preparation method according to item 1, characterized in that the molecular weight of sodium hyaluronate is 3kDa-2200kDa.

4.根据项1所述的制备方法，其特征在于，透明质酸钠与钙盐的质量比为1:0.5～5，优选为1:1～3。4. The preparation method according to item 1, characterized in that the mass ratio of sodium hyaluronate to calcium salt is 1:0.5-5, preferably 1:1-3.

5.根据项1所述的制备方法，其特征在于，所述置换液和所述洗涤液的pH分别为5.0～7.0，所述洗涤液中有机介质浓度为50％～90wt％，优选为60％～80wt％，5. The preparation method according to item 1, characterized in that, the pH of the replacement liquid and the washing liquid are respectively 5.0 to 7.0, and the concentration of the organic medium in the washing liquid is 50% to 90 wt%, preferably 60 %～80wt%,

所述脱水液中有机介质浓度大于等于90wt％。The concentration of the organic medium in the dehydration liquid is greater than or equal to 90wt%.

6.根据项1所述的制备方法，其特征在于，置换的次数为1～4次，优选为1～2次，置换时间为1～24h，优选为5～16h；优选地，洗涤次数为1～5次，优选为2～3次；进一步优选地，脱水次数为1～5次，优选为2～3次。6. The preparation method according to item 1, characterized in that the number of replacements is 1 to 4 times, preferably 1 to 2 times, and the replacement time is 1 to 24 hours, preferably 5 to 16 hours; preferably, the number of times of washing is 1 to 5 times, preferably 2 to 3 times; more preferably, the dehydration times are 1 to 5 times, preferably 2 to 3 times.

7.根据项1所述的制备方法，其特征在于，所述钙盐选自氯化钙、硝酸钙、乙酸钙、甲酸钙、亚硝酸钙中的一种或两种以上。7. The preparation method according to item 1, wherein the calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium acetate, calcium formate, and calcium nitrite.

8.根据项1所述的制备方法，其特征在于，所得到的透明质酸钙粉末中透明质酸钙的含量大于等于90wt％，在1wt％水溶液中的透光率大于等于99％。8. The preparation method according to item 1, characterized in that the calcium hyaluronate content in the obtained calcium hyaluronate powder is greater than or equal to 90 wt%, and the light transmittance in a 1 wt% aqueous solution is greater than or equal to 99%.

9.一种透明质酸钙，其特征在于，所述透明质酸钙的分子量为2kDa-2000kDa，优选为2kDa-1000kDa。9. A calcium hyaluronate, characterized in that the molecular weight of the calcium hyaluronate is 2kDa-2000kDa, preferably 2kDa-1000kDa.

10.根据项9所述的透明质酸钙，其特征在于，在1wt％水溶液中所述透明质酸钙的透光率大于等于99％。10. The calcium hyaluronate according to item 9, characterized in that the light transmittance of the calcium hyaluronate in a 1wt% aqueous solution is greater than or equal to 99%.

11.根据项9或10所述的透明质酸钙，其特征在于，所述透明质酸钙通过将透明质酸钠加入到含有钙盐的酸性置换液中进行置换得到，所述置换液中有机介质浓度为60％～80wt％，钙盐浓度为1％～3wt％，所述置换液pH为5.0～7.0。11. The calcium hyaluronate according to item 9 or 10, characterized in that, the calcium hyaluronate is obtained by adding sodium hyaluronate to an acidic replacement solution containing calcium salts for replacement, and the replacement solution The concentration of the organic medium is 60%-80wt%, the concentration of the calcium salt is 1%-3wt%, and the pH of the replacement solution is 5.0-7.0.

12.项1-8中任一项所述的制备方法制备得到的透明质酸钙，或项9-11中任一项所述的透明质酸钙，在损伤修复、提高皮肤角质层含水量、减少皮肤细纹、抗氧化中的应用。12. The calcium hyaluronate prepared by the preparation method described in any one of items 1-8, or the calcium hyaluronate described in any one of items 9-11, is effective in repairing damage and increasing the water content of the stratum corneum of the skin , reduce skin fine lines, anti-oxidation in the application.

13.一种损伤修复、提高皮肤角质层含水量、减少皮肤细纹、抗氧化的方法，其包括向受试者施用项1-8中任一项所述的制备方法制备得到的透明质酸钙，或项9-11中任一项所述的透明质酸钙。13. A method for repairing damage, increasing the water content of the stratum corneum of the skin, reducing fine lines of the skin, and anti-oxidation, which comprises applying hyaluronic acid prepared by the preparation method described in any one of items 1-8 to the subject Calcium, or calcium hyaluronate according to any one of items 9-11.

14.一种组合物，其包括项9-11中任一项所述的透明质酸钙。14. A composition comprising calcium hyaluronate according to any one of items 9-11.

本申请通过高效率的置换，得到透明质酸钙，解决了目前透明质酸钙生产工艺中置换次数多、耗时长、钙盐消耗大、钙置换效率低、透明质酸钙溶液混浊的问题。具体地，本申请制备透明质酸钙的收率在93.75％以上，透光率在98.5％以上。The present application obtains calcium hyaluronate through high-efficiency replacement, which solves the problems of many replacement times, long time consumption, large calcium salt consumption, low calcium replacement efficiency, and cloudy calcium hyaluronate solution in the current production process of calcium hyaluronate. Specifically, the yield of calcium hyaluronate prepared by the present application is above 93.75%, and the light transmittance is above 98.5%.

附图说明Description of drawings

图1显示不同分子量透明质酸钙样品的羟自由基清除率；Fig. 1 shows the hydroxyl radical scavenging rate of different molecular weight calcium hyaluronate samples;

图2显示不同浓度及分子量透明质酸钙样品的羟自由基清除率；Figure 2 shows the hydroxyl radical scavenging rate of different concentrations and molecular weight calcium hyaluronate samples;

图3显示不同分子量透明质酸钙样品的超氧阴离子自由基清除率；Fig. 3 shows the superoxide anion radical scavenging rate of different molecular weight calcium hyaluronate samples;

图4显示不同浓度及分子量透明质酸钙样品的超氧阴离子自由基清除率。Figure 4 shows the superoxide anion radical scavenging rate of calcium hyaluronate samples with different concentrations and molecular weights.

具体实施方式Detailed ways

下面结合实施例进一步说明本申请，应当理解，实施例仅用于进一步说明和阐释本申请，并非用于限制本申请。The present application will be further described below in conjunction with the examples. It should be understood that the examples are only used to further illustrate and explain the present application, and are not intended to limit the present application.

除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。以下结合具体实施例对本申请作进一步的说明，但不用来限制本申请的范围。Unless otherwise defined, technical and scientific terms in this specification have the same meaning as commonly understood by a person skilled in the art. Although methods and materials similar or identical to those described herein can be employed in experiments or practical applications, the materials and methods are described herein below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are presented for purposes of illustration only and not limitation. The present application will be further described below in conjunction with specific examples, but they are not used to limit the scope of the present application.

本申请提供一种透明质酸钙的制备方法，其特征在于，包括以下步骤：The application provides a preparation method of calcium hyaluronate, characterized in that, comprising the following steps:

将洗涤后的所述沉淀用中性脱水液进行脱水，真空干燥，得到透明质酸钙粉末。The washed precipitate is dehydrated with a neutral dehydrating solution, and vacuum-dried to obtain calcium hyaluronate powder.

其中含有钙盐的酸性置换液为经过有机滤膜过滤得到的，其中滤膜孔径要求0.2～3微米，优选0.22～1.2微米。The acidic replacement solution containing calcium salt is obtained by filtering through an organic filter membrane, wherein the pore diameter of the filter membrane is required to be 0.2-3 microns, preferably 0.22-1.2 microns.

置换液为酸性，包括钙盐、有机介质和水。置换液的pH可以采用不影响置换的酸进行调节，例如冰醋酸等。其中所述置换液中有机介质浓度为50％～90wt％，例如可以为50wt％、55wt％、60wt％、65wt％、70wt％、75wt％、80wt％、85wt％、90wt％，优选为60％～80wt％。钙盐浓度为0.5％～4wt％，例如可以为0.5wt％、1wt％、1.5wt％、2wt％、2.5wt％、3wt％、3.5wt％、4wt％，优选为1％～3wt％。The replacement fluid is acidic and includes calcium salts, organic media and water. The pH of the replacement solution can be adjusted with an acid that does not affect the replacement, such as glacial acetic acid. Wherein the organic medium concentration in the replacement liquid is 50% to 90wt%, such as 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%, 80wt%, 85wt%, 90wt%, preferably 60% ~80wt%. The calcium salt concentration is 0.5%-4wt%, such as 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, preferably 1%-3wt%.

其中的有机介质为与水相容性好、但透明质酸钠或透明质酸钙在其中不溶或微溶的有机介质，优选为醇类有机溶剂或酮类有机溶剂，更优选为乙醇、甲醇或丙酮。The organic medium is an organic medium with good water compatibility but insoluble or slightly soluble in sodium hyaluronate or calcium hyaluronate, preferably alcohol organic solvent or ketone organic solvent, more preferably ethanol, methanol or acetone.

所述钙盐选自氯化钙、硝酸钙、乙酸钙、甲酸钙、亚硝酸钙中的一种或两种以上。The calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium acetate, calcium formate, and calcium nitrite.

置换步骤中透明质酸钠的分子量可以根据所需制备的透明质酸钙的分子量进行调整。在一个具体的实施方式中，所述透明质酸钠的分子量为3kDa～2200kDa，例如可以为3kDa、10kDa、50kDa、100kDa、200kDa、300kDa、400kDa、500kDa、600kDa、700kDa、800kDa、900kDa、1000kDa、1100kDa、1200kDa、1300kDa、1400kDa、1500kDa、1600kDa、1700kDa、1800kDa、1900kDa、2000kDa、2100kDa、2200kD等。The molecular weight of sodium hyaluronate in the replacement step can be adjusted according to the molecular weight of calcium hyaluronate to be prepared. In a specific embodiment, the molecular weight of the sodium hyaluronate is 3kDa-2200kDa, for example, it can be 3kDa, 10kDa, 50kDa, 100kDa, 200kDa, 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa, 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, etc.

在一个具体的实施方式中，所述有机介质为乙醇。In a specific embodiment, the organic medium is ethanol.

在一个具体的实施方式中，所述置换液的pH为5.0～7.0，例如可以为5.0、5.5、6.0、6.5、6.8、6.9等。In a specific embodiment, the pH of the replacement solution is 5.0-7.0, such as 5.0, 5.5, 6.0, 6.5, 6.8, 6.9, etc.

进行置换时通常在搅拌下进行置换。置换的次数和时间可以根据情况进行调整。在一个具体的实施方式中，置换的次数为1～4次，例如可以为1次、2次、3次、4次，优选为1～2次。置换时间为1～24h，例如可以为1h、2h、5h、8h、10h、12h、15h、18h、20h、24h，优选为5～16h。The substitution is usually performed under stirring. The number and time of replacement can be adjusted according to the situation. In a specific embodiment, the number of substitutions is 1 to 4 times, for example, 1 time, 2 times, 3 times, 4 times, preferably 1 to 2 times. The replacement time is 1-24h, for example, 1h, 2h, 5h, 8h, 10h, 12h, 15h, 18h, 20h, 24h, preferably 5-16h.

洗涤液为酸性，包括有机介质和水。洗涤液的pH可以采用不影响洗涤的酸进行调节，例如冰醋酸等。所述洗涤液中有机介质浓度为50％～90wt％，例如可以为50wt％、55wt％、60wt％、65wt％、70wt％、75wt％、80wt％、85wt％、90wt％，优选为60％～80wt％。The washing liquid is acidic, including organic medium and water. The pH of the washing solution can be adjusted with an acid that does not affect washing, such as glacial acetic acid. The concentration of the organic medium in the washing liquid is 50% to 90wt%, such as 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%, 80wt%, 85wt%, 90wt%, preferably 60% to 80 wt%.

有机介质如上所述，在一个具体的实施方式中，所述有机介质为乙醇。Organic medium As mentioned above, in a specific embodiment, the organic medium is ethanol.

在一个具体的实施方式中，所述洗涤液的pH为5.0～7.0例如可以为5.0、5.5、6.0、6.5、6.8、6.9等。In a specific embodiment, the pH of the washing solution is 5.0-7.0, such as 5.0, 5.5, 6.0, 6.5, 6.8, 6.9, etc.

洗涤液与透明质酸钙沉淀的体积比为1～3:1，例如可以为1:1、2:1、3:1，优选1～2:1。The volume ratio of the washing solution to the calcium hyaluronate precipitate is 1-3:1, for example, 1:1, 2:1, 3:1, preferably 1-2:1.

洗涤的次数可以根据情况进行调整。在一个具体的实施方式中，洗涤的次数为1～5次，例如可以为1次、2次、3次、4次、5次，优选为2～3次。The number of times of washing can be adjusted according to the situation. In a specific embodiment, the number of times of washing is 1 to 5 times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, preferably 2 to 3 times.

脱水液为中性，包括有机介质和水。所述脱水液中有机介质浓度大于等于90wt％，例如可以为90wt％、91wt％、92wt％、93wt％、94wt％、95wt％、96wt％、97wt％、98wt％、99wt％。The dehydration liquid is neutral, including organic medium and water. The concentration of the organic medium in the dehydration liquid is greater than or equal to 90wt%, such as 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, 96wt%, 97wt%, 98wt%, 99wt%.

脱水的次数可以根据情况进行调整。在一个具体的实施方式中，脱水的次数为1～5次，例如可以为1次、2次、3次、4次、5次，优选为2～3次。The number of times of dehydration can be adjusted according to the situation. In a specific embodiment, the number of times of dehydration is 1 to 5 times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, preferably 2 to 3 times.

在一个具体的实施方式中，透明质酸钙的制备方法包括以下步骤：In a specific embodiment, the preparation method of calcium hyaluronate comprises the following steps:

将透明质酸钠加入到含有钙盐的酸性置换液中置换1～4次，置换时间为1～24h，得到透明质酸钙沉淀，其中所述置换液还包括有机介质和水，所述置换液中有机介质浓度为50％～90wt％，钙盐浓度为0.5％～4wt％，所述置换液的pH为5.0～7.0；adding sodium hyaluronate into an acidic replacement solution containing calcium salt and replacing it for 1 to 4 times, and the replacement time is 1 to 24 hours to obtain a calcium hyaluronate precipitate, wherein the replacement solution also includes an organic medium and water, and the replacement The concentration of the organic medium in the solution is 50%-90wt%, the concentration of calcium salt is 0.5%-4wt%, and the pH of the replacement solution is 5.0-7.0;

用酸性洗涤液洗涤所述沉淀1～5次，其中所述酸性洗涤液包括有机介质和水，所述洗涤液中有机介质浓度为50％～90wt％，所述洗涤液与透明质酸钙沉淀的体积比为1～3:1；Washing the precipitate with an acidic washing liquid for 1 to 5 times, wherein the acidic washing liquid includes an organic medium and water, the concentration of the organic medium in the washing liquid is 50% to 90 wt%, and the washing liquid and the calcium hyaluronate precipitate The volume ratio is 1 to 3:1;

将洗涤后的所述沉淀用中性脱水液脱水1～5次，真空干燥，得到透明质酸钙粉末，其中所述脱水液包括有机介质和水，所述脱水液中有机介质浓度大于等于90wt％。Dehydrate the washed precipitate with a neutral dehydration solution for 1 to 5 times, and dry it in vacuum to obtain calcium hyaluronate powder, wherein the dehydration solution includes an organic medium and water, and the concentration of the organic medium in the dehydration solution is greater than or equal to 90wt %.

将透明质酸钠加入到含有钙盐的酸性置换液中置换1～2次，置换时间为5～16h，得到透明质酸钙沉淀，其中所述置换液还包括有机介质和水，所述置换液中有机介质浓度为60％～80wt％，钙盐浓度为1％～3wt％，所述置换液的pH为5.0～7.0；Adding sodium hyaluronate into an acidic replacement solution containing calcium salts for replacement 1-2 times, the replacement time is 5-16 hours, to obtain a calcium hyaluronate precipitate, wherein the replacement solution also includes an organic medium and water, and the replacement The concentration of the organic medium in the solution is 60%-80wt%, the concentration of calcium salt is 1%-3wt%, and the pH of the replacement solution is 5.0-7.0;

用酸性洗涤液洗涤所述沉淀2～3次，其中所述酸性洗涤液包括有机介质和水，所述洗涤液中有机介质浓度为50％～90wt％，所述洗涤液与透明质酸钙沉淀的体积比为1～2:1；Wash the precipitate 2 to 3 times with an acidic washing liquid, wherein the acidic washing liquid includes an organic medium and water, the concentration of the organic medium in the washing liquid is 50% to 90 wt%, and the washing liquid and the calcium hyaluronate precipitate The volume ratio is 1～2:1;

将洗涤后的所述沉淀用中性脱水液脱水2～3次，真空干燥，得到透明质酸钙粉末，其中所述脱水液包括有机介质和水，所述脱水液中有机介质浓度大于等于90wt％。The washed precipitate is dehydrated 2 to 3 times with a neutral dehydration solution, and vacuum-dried to obtain calcium hyaluronate powder, wherein the dehydration solution includes an organic medium and water, and the concentration of the organic medium in the dehydration solution is greater than or equal to 90wt %.

将洗涤后的所述沉淀用中性脱水液脱水2～3次，真空干燥，得到透明质酸钙粉末，其中所述脱水液包括有机介质和水，所述脱水液中有机介质浓度大于等于90wt％；The washed precipitate is dehydrated 2 to 3 times with a neutral dehydration solution, and vacuum-dried to obtain calcium hyaluronate powder, wherein the dehydration solution includes an organic medium and water, and the concentration of the organic medium in the dehydration solution is greater than or equal to 90wt %;

其中所述有机介质为乙醇。Wherein said organic medium is ethanol.

在一个具体的实施方式中，所得到的透明质酸钙中钙离子含量为3.0～10.0wt％，例如可以为3wt％、4wt％、5wt％、6wt％、7wt％、8wt％、9wt％、10wt％。In a specific embodiment, the calcium ion content in the obtained calcium hyaluronate is 3.0-10.0wt%, for example, it can be 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10 wt%.

在一个具体的实施方式中，所得到的透明质酸钙在1wt％水溶液中的透光率大于等于99％，例如为99％、99.1％、99.2％、99.3％、99.4％、99.5％，99.6％，99.7％，99.8，99.9％等。In a specific embodiment, the light transmittance of the obtained calcium hyaluronate in a 1wt% aqueous solution is greater than or equal to 99%, such as 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% %, 99.7%, 99.8, 99.9%, etc.

在一个具体的实施方式中，所得到的透明质酸钙中透明质酸钙的含量大于等于90wt％，例如为90wt％、91wt％、92wt％、93wt％、94wt％、95wt％、96wt％、97wt％、98wt％、99wt％等。In a specific embodiment, the content of calcium hyaluronate in the obtained calcium hyaluronate is greater than or equal to 90wt%, such as 90wt%, 91wt%, 92wt%, 93wt%, 94wt%, 95wt%, 96wt%, 97wt%, 98wt%, 99wt%, etc.

在一个具体的实施方式中，所得到的透明质酸钙中钠离子的含量下小于等于3％，例如为0.2％、0.4％、0.6％、0.8％、1.0％、1.2％、1.4％、1.8％、2.0％、2.8％、3.0％等。In a specific embodiment, the sodium ion content in the obtained calcium hyaluronate is less than or equal to 3%, such as 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.8% %, 2.0%, 2.8%, 3.0%, etc.

在一个具体的实施方式中，透明质酸钙采用上述方法制备得到。In a specific embodiment, calcium hyaluronate is prepared by the above method.

在本申请中，所得到的透明质酸钙分子量的测定方法为特性黏数法。具体测定方法如下。In the present application, the method for measuring the molecular weight of the obtained calcium hyaluronate is the intrinsic viscosity method. The specific measurement method is as follows.

供试液制备：精密称供试品约100mg(W ₁)(注：该称样量不确定，当特性黏数低于3.0dL/g或分子量低于10万时应增加称样量)至100ml容量瓶中，加0.2mol/L氯化纳溶液定容，摇匀；再称取该溶液一定量(W ₂)(注:该称样量不确定，根据测定的供试液的流出时间调整)至100ml容量瓶中，加0.2mo1/L氯化钠溶液定容，摇匀，作为供试液。 Preparation of the test solution: Accurately weigh about 100 mg (W ₁ ) of the test product (Note: The sample size is uncertain, and the sample size should be increased when the intrinsic viscosity is lower than 3.0 dL/g or the molecular weight is lower than 100,000) to In a 100ml volumetric flask, add 0.2mol/L sodium chloride solution to constant volume, shake up; then take a certain amount of this solution (W ₂ ) (note: the weighing sample size is uncertain, according to the outflow time of the test solution measured) Adjust) to a 100ml volumetric flask, add 0.2mol/L sodium chloride solution to constant volume, shake well, and use it as the test solution.

溶剂流出时间(t ₀)的测定：取滤过的0.2mol/L氯化钠溶液适量，弃去初滤液，取续滤液(不得少于7ml)注入洁净、干燥的乌氏黏度计，将黏度计垂直固定于恒温水浴(温度25℃土0.1℃)中，放置15分钟，准确记录流出时间，重复测定至少两次，取平均值为空白液的流出时间(t ₀)。 Determination of solvent outflow time (t ₀ ): Take an appropriate amount of filtered 0.2mol/L sodium chloride solution, discard the initial filtrate, take the subsequent filtrate (not less than 7ml) and inject it into a clean and dry Ubbelohde viscometer, and measure the viscosity Fix the meter vertically in a constant temperature water bath (temperature 25°C ± 0.1°C), place it for 15 minutes, accurately record the outflow time, repeat the measurement at least twice, and take the average value as the outflow time of the blank solution (t ₀ ).

供试液流出时间(t ₁)的测定：取供试液适量，按照溶剂流出时间的测定方法进行测定，重复测定至少两次，两次测定值相差不得超过0.2秒，取平均值为供试液的流出时间(t ₁)。 Determination of the outflow time ( _t1 ) of the test solution: take an appropriate amount of the test solution, measure it according to the method for measuring the outflow time of the solvent, repeat the measurement at least twice, and the difference between the two measured values shall not exceed 0.2 seconds, and take the average value as the test solution. The outflow time of the liquid (t ₁ ).

t ₁/t ₀应在1.3～1.5之间，否则应调整供试液浓度后再测。 t ₁ /t ₀ should be between 1.3 and 1.5, otherwise the concentration of the test solution should be adjusted before testing.

采用下式计算供试液浓度C(g/dL)：Use the following formula to calculate the concentration C (g/dL) of the test solution:

ρ ₂₅＝1.000g/ml(供试液在25℃的密度) ρ ₂₅ =1.000g/ml (the density of the test solution at 25°C)

采用下式计算特性黏数：The intrinsic viscosity was calculated using the following formula:

计算透明质酸钙平均分子量：Calculate the average molecular weight of calcium hyaluronate:

特性粘度[η](dL/g)＝3.6×10 ^-4M ^0.78(M：平均分子量) Intrinsic viscosity [η] (dL/g) = 3.6×10 ^-4 M ^0.78 (M: average molecular weight)

在本申请中，所得到的透明质酸钙的透光率的测定方法为光吸收法。具体测定方法如下。In the present application, the method for measuring the light transmittance of the obtained calcium hyaluronate is the light absorption method. The specific measurement method is as follows.

取样品0.1g至盛有100ml水的锥形瓶中，在冰箱中放置过夜，溶解后，将样品溶液倒入1ml比色皿中，以水作空白，在550nm波长处测定溶液的透光率。Take 0.1g of the sample into a conical flask filled with 100ml of water, place it in the refrigerator overnight, after dissolving, pour the sample solution into a 1ml cuvette, use water as a blank, and measure the light transmittance of the solution at a wavelength of 550nm .

在本申请中，所得到的透明质酸钙的钙含量的测定方法为火焰原子吸收法。具体测定方法如下。In the present application, the method for measuring the calcium content of the obtained calcium hyaluronate is the flame atomic absorption method. The specific measurement method is as follows.

标准曲线制备：Standard curve preparation:

精密称取钙单元素溶液标准物质(1000μg/ml)1ml至100ml容量瓶中，用1％硝酸定容，得标准贮备液(10μg/m1)。分别量取标准贮备液0、0.2、0.4、0.8、1.6ml于5个洁净的10ml容量瓶中，用1％硝酸定容。得钙浓度分别为0、0.2、0.4、0.8、1.6μg/m1的钙标准溶液。Accurately weigh 1ml of calcium single element solution standard substance (1000μg/ml) into a 100ml volumetric flask, and dilute to volume with 1% nitric acid to obtain a standard stock solution (10μg/ml). Measure standard stock solution 0, 0.2, 0.4, 0.8, 1.6ml respectively into five clean 10ml volumetric flasks, and dilute to volume with 1% nitric acid. Calcium standard solutions with calcium concentrations of 0, 0.2, 0.4, 0.8, and 1.6 μg/m1 were obtained.

供试品溶液：精密称取供试品50mg至100ml容量瓶中，加入硝酸1ml，水浴加热至完全浴解，用水定容，取上述溶液1ml至50ml容量瓶中，用1％硝酸定容，即得。Test solution: Accurately weigh 50 mg of the test product into a 100ml volumetric flask, add 1ml of nitric acid, heat in a water bath until completely dissolved, and dilute with water. Take 1ml of the above solution into a 50ml volumetric flask, and dilute with 1% nitric acid. Instantly.

测定：用空白溶液调零后，取各溶液适量，以火焰原子吸收法测定，检测波长为213.9nm，测定吸光度。以测试溶液吸光度(Y)对钙的浓度(X，μg/m1)进行线性回归，绘制标准曲线，采用下式计算供试液中钙浓度。Determination: After adjusting to zero with a blank solution, take an appropriate amount of each solution, and measure it by flame atomic absorption method. The detection wavelength is 213.9nm, and the absorbance is measured. Carry out linear regression with the absorbance (Y) of the test solution against the concentration of calcium (X, μg/m1), draw a standard curve, and use the following formula to calculate the calcium concentration in the test solution.

式中，C _i—供试液中钙浓度，μg/m1； In the formula, C _i —calcium concentration in the test solution, μg/m1;

W—供试品称样量，mg；W—the weighing amount of the test product, mg;

h％—供试品干燥失重。h%—the loss on drying of the test product.

在本申请中，糖醛酸含量采用硫酸-咔唑比色法进行检测。In this application, the content of uronic acid is detected by the sulfuric acid-carbazole colorimetric method.

本申请中，收率为出料量与投料量之比，计算公式为：In the present application, the yield is the ratio of the amount of output to the amount of feed, and the calculation formula is:

收率＝透明质酸钙出料量/透明质酸钠投料量×100％Yield = calcium hyaluronate output/sodium hyaluronate input × 100%

本申请还提供一种透明质酸钙，所述透明质酸钙的分子量为2kDa-2000kDa，例如可以为2kDa、10kDa、20kDa、50kDa、100kDa、200kDa、300kDa、400kDa、500kDa、600kDa、700kDa、800kDa、900kDa、1000kDa、1100kDa、1200kDa、1300kDa、1400kDa、1500kDa、1600kDa、1700kDa、1800kDa、1900kDa、2000kDa。The present application also provides a calcium hyaluronate, the molecular weight of the calcium hyaluronate is 2kDa-2000kDa, such as 2kDa, 10kDa, 20kDa, 50kDa, 100kDa, 200kDa, 300kDa, 400kDa, 500kDa, 600kDa, 700kDa, 800kDa , 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa.

在一个具体的实施方式中，在1wt％水溶液中所述透明质酸钙的透光率大于等于99％。In a specific embodiment, the light transmittance of the calcium hyaluronate in a 1 wt% aqueous solution is greater than or equal to 99%.

在一个具体的实施方式中，所述透明质酸钙通过将透明质酸钠加入到含有钙盐的酸性置换液中进行置换得到，所述置换液中有机介质浓度为60％～80wt％，钙盐浓度为1％～3wt％，所述置换液pH为5.0～7.0。In a specific embodiment, the calcium hyaluronate is obtained by adding sodium hyaluronate into an acidic replacement solution containing calcium salts for replacement, the concentration of the organic medium in the replacement solution is 60% to 80% by weight, calcium The salt concentration is 1%-3wt%, and the pH of the replacement solution is 5.0-7.0.

在一个具体的实施方式中，所述透明质酸钙通过本申请上述制备方法制备得到。In a specific embodiment, the calcium hyaluronate is prepared by the above-mentioned preparation method of the present application.

所述透明质酸钙具有透明质酸的各种功能，例如可以用于损伤修复、提高皮肤角质层含水量、减少皮肤细纹、抗氧化。The calcium hyaluronate has various functions of hyaluronic acid, for example, it can be used for repairing damage, increasing the water content of the stratum corneum of the skin, reducing fine lines of the skin, and anti-oxidation.

本申请还提供上述任意一种方法制备的透明质酸钙或上述的任意一种透明质酸钙在损伤修复、提高皮肤角质层含水量、减少皮肤细纹、抗氧化中的应用。The present application also provides the calcium hyaluronate prepared by any one of the above methods or the application of any one of the above calcium hyaluronates in repairing damage, increasing the water content of the stratum corneum of the skin, reducing fine lines of the skin, and anti-oxidation.

本申请还提供一种损伤修复的方法，其包括向受试者上述任意一种方法制备的透明质酸钙或上述的任意一种透明质酸钙。The present application also provides a method for repairing damage, which comprises administering calcium hyaluronate or any calcium hyaluronate prepared by any of the above-mentioned methods to a subject.

本申请还提供一种提高皮肤角质层含水量的方法，其包括向受试者上述任意一种方法制备的透明质酸钙或上述的任意一种透明质酸钙。The present application also provides a method for increasing the water content of the stratum corneum of the skin, which comprises administering calcium hyaluronate prepared by any one of the above-mentioned methods or any one of the above-mentioned calcium hyaluronates to the subject.

本申请还提供一种减少皮肤细纹的方法，其包括向受试者上述任意一种方法制备的透明质酸钙或上述的任意一种透明质酸钙。The present application also provides a method for reducing skin fine lines, which comprises administering calcium hyaluronate prepared by any one of the above methods or any one of the above calcium hyaluronates to a subject.

本申请还提供一种抗氧化的方法，其包括向受试者上述任意一种方法制备的透明质酸钙或上述的任意一种透明质酸钙。The present application also provides an anti-oxidation method, which comprises administering calcium hyaluronate or any calcium hyaluronate prepared by any one of the above-mentioned methods to a subject.

本申请还提供一种含有上述透明质酸钙的组合物。The present application also provides a composition containing the above-mentioned calcium hyaluronate.

实施例1Example 1

配制4.0wt％氯化钙-乙醇(酸性)置换液2.5T，氯化钙100kg，使用冰乙酸调节乙醇pH为5.6，置换液浓度为80wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 2.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution, 100kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 5.6, and the concentration of the replacement solution to be 80wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量1480kDa)投入到置换液中，开启搅拌进行置换，置换时间为24h。一次置换完成后，停止搅拌静置约1-2h，抽掉多余上清，共计置换1次，每次约24h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 1480kDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 24h. After one replacement is completed, stop stirring and let stand for about 1-2 hours, remove excess supernatant, and replace once in total, about 24 hours each time.

置换完成后，静置后将上清抽出，加入与沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为78％、pH为5.6，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and washing liquid was added with a volume equal to the volume of the precipitate for washing. The ethanol concentration of the washing liquid was 78%, the pH was 5.6, and the washing frequency was 1 time, and the washing time was 3 hours.

洗涤完成后将多余上清抽出，加入与沉淀体积等倍体积的浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为20h，出料得到透明质酸钙19.4kg。After washing, extract excess supernatant, add 90% ethanol dehydration liquid with a concentration equal to the volume of the precipitate for dehydration, dehydration time is 0.5-1 hour, dehydration 3 times. Ensure that the alcohol content of the supernatant is above 90%, and then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, and the drying time is 20h, and 19.4kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为7.2％，钠含量为0.23％，收率为97％，分子量为127万，0.1％透明质酸钙溶液的透光率为99.5％，糖醛酸含量48.16％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 7.2%, the sodium content is 0.23%, the yield is 97%, the molecular weight is 1.27 million, and the light transmittance of 0.1% calcium hyaluronate solution is 99.5%. The aldehyde acid content is 48.16%.

实施例2Example 2

配制4.0wt％氯化钙-乙醇(酸性)置换液1.5T，氯化钙60kg使用冰乙酸调节乙醇pH为6.2，置换液浓度为80wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution, use glacial acetic acid to adjust the pH of ethanol to 6.2 with 60 kg of calcium chloride, and the concentration of the replacement solution is 80 wt%. Use a 0.45 micron polypropylene filter before use.

置换完成后，静置后将上清抽出，加入与沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为75％、pH为5.8，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and washing liquid was added in a volume equal to the volume of the precipitate for washing. The ethanol concentration of the washing liquid was 75%, the pH was 5.8, and the washing frequency was 1 time, and the washing time was 3 hours.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积的浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为22h，出料得到透明质酸钙19.2kg。After the washing is completed, the excess supernatant is extracted, and an ethanol dehydration solution with a concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is more than 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum is 0.10MPa, the drying time is 22h, and the discharge obtains 19.2kg of calcium hyaluronate.

检测样品指标得出透明质酸钙的钙离子含量为6.8％，钠含量为0.35％，收率为96％，分子量为130万，0.1％透明质酸钙溶液的透光率为99.3％，糖醛酸含量47.93％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 6.8%, the sodium content is 0.35%, the yield is 96%, the molecular weight is 1.3 million, the light transmittance of 0.1% calcium hyaluronate solution is 99.3%, sugar The aldehyde acid content is 47.93%.

实施例3Example 3

配制4.0wt％氯化钙-乙醇(酸性)置换液0.5T，氯化钙20kg，使用冰乙酸调节乙醇pH为6.0，置换液浓度为80wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 0.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution, 20kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 6.0, and the concentration of the replacement solution to be 80wt%. Use a 0.45 micron polypropylene filter before use.

置换完成后，静置后将上清抽出，加入与沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为78％、pH为6.20，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing solution equal to the volume of the precipitation was added for washing. The ethanol concentration of the washing solution was 78%, the pH was 6.20, and the washing frequency was 1 time, and the washing time was 3 hours each time.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积的浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为22h，出料得到透明质酸钙19.27kg。After the washing is completed, the excess supernatant is extracted, and an ethanol dehydration solution with a concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, and 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, and the drying time is 22 hours. The output is 19.27 kg of calcium hyaluronate.

检测样品指标得出透明质酸钙的钙离子含量为5.0％，钠含量为0.54％，收率为96.35％，分子量为132万，0.1％透明质酸钙溶液的透光率为99.4％，糖醛酸含量48.54％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 5.0%, the sodium content is 0.54%, the yield is 96.35%, the molecular weight is 1.32 million, the light transmittance of 0.1% calcium hyaluronate solution is 99.4%, sugar The aldehyde acid content is 48.54%.

实施例4Example 4

配制4.0wt％氯化钙-乙醇(酸性)置换液0.25T，氯化钙20Kg，使用冰乙酸调节乙醇pH为5.8，置换液浓度为75wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 0.25T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution, 20Kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 5.8, and the concentration of the replacement solution to be 75wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量1360KDa)投入到置换液中，开启搅拌进行置换，置换时间为24h。一次置换完成后，停止搅拌静置约1-2h，抽掉多余上清，共计置换1次，每次约24h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 1360KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 24h. After one replacement is completed, stop stirring and let stand for about 1-2 hours, remove excess supernatant, and replace once in total, about 24 hours each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为72％、pH为6.2，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 72%, and the pH was 6.2.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为23h，出料得到透明质酸钙19.30kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 23h, and 19.30kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为4.8％，钠含量为0.62％，收率为96.51％，分子量为115万，0.1％透明质酸钙溶液的透光率为99.2％，糖醛酸含量48.32％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 4.8%, the sodium content is 0.62%, the yield is 96.51%, the molecular weight is 1.15 million, the light transmittance of 0.1% calcium hyaluronate solution is 99.2%, sugar The aldehyde acid content is 48.32%.

实施例5Example 5

配制3.0wt％氯化钙-乙醇(酸性)置换液1.8T，氯化钙60Kg，使用冰乙酸调节乙醇pH为6.5，置换液浓度为80wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.8T of 3.0wt% calcium chloride-ethanol (acidic) replacement solution, 60Kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 6.5, and the concentration of the replacement solution is 80wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量320KDa)投入到置换液中，开启搅拌进行置换，置换时间为24h。一次置换完成后，停止搅拌静置约1-2h，抽掉多余上清，共计置换1次，每次约24h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight: 320KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 24 hours. After one replacement is completed, stop stirring and let stand for about 1-2 hours, remove excess supernatant, and replace once in total, about 24 hours each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为75％、pH为6.32，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 75%, the pH was 6.32, and the washing frequency was 1 time, and the washing time was 3 hours.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为20h，出料得到透明质酸钙19.34kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 20h, and 19.34kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为5.6％，钠含量为0.32％，收率为96.7％，分子量为29万，0.1％透明质酸钙溶液的透光率为99.7％，糖醛酸含量48.33％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 5.6%, the sodium content is 0.32%, the yield is 96.7%, the molecular weight is 290,000, the light transmittance of 0.1% calcium hyaluronate solution is 99.7%, sugar The aldehyde acid content is 48.33%.

实施例6Example 6

配制3.0wt％乙酸钙-乙醇(酸性)置换液1.8T，乙酸钙60Kg，使用冰乙酸调节乙醇pH为6.5，置换液浓度为80wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.8T of 3.0wt% calcium acetate-ethanol (acidic) replacement solution, 60Kg of calcium acetate, use glacial acetic acid to adjust the pH of ethanol to 6.5, and the concentration of the replacement solution is 80wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量320KDa)投入到置换液中，开启搅拌进行置换，置换时间为16h。一次置换完成后，停止搅拌静置约1-2h，抽掉多余上清，共计置换1次，每次约16h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 320KDa) and put it into the replacement solution, start stirring to replace, and the replacement time is 16h. After one replacement is completed, stop stirring and let it stand for about 1-2 hours, remove excess supernatant, and replace once in total, about 16 hours each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为78％、pH为5.82，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 78%, the pH was 5.82, and the washing frequency was 1 time, and the washing time was 3 hours.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为22h，出料得到透明质酸钙19.28kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 22h, and 19.28kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为5.5％，钠含量为0.35％，收率为96.4％，分子量为27万，0.1％透明质酸钙溶液的透光率为99.5％，糖醛酸含量48.41％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 5.5%, the sodium content is 0.35%, the yield is 96.4%, the molecular weight is 270,000, the light transmittance of 0.1% calcium hyaluronate solution is 99.5%, sugar The aldehyde acid content is 48.41%.

实施例7Example 7

配制4.0wt％硝酸钙-乙醇(酸性)置换液2.0T，硝酸钙60Kg，使用冰乙酸调节乙醇pH为6.8，置换液浓度为70wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 2.0T of 4.0wt% calcium nitrate-ethanol (acidic) replacement solution, 60Kg of calcium nitrate, use glacial acetic acid to adjust the pH of ethanol to 6.8, and the concentration of the replacement solution is 70wt%. Use a 0.45 micron polypropylene filter before use.

检测样品指标得出透明质酸钙的钙离子含量为6.2％，钠含量为0.34％，收率为96.4％，分子量为25万，0.1％透明质酸钙溶液的透光率为99.5％，糖醛酸含量48.76％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 6.2%, the sodium content is 0.34%, the yield is 96.4%, the molecular weight is 250,000, the light transmittance of 0.1% calcium hyaluronate solution is 99.5%, sugar The aldehyde acid content is 48.76%.

实施例8Example 8

配制2.0wt％氯化钙-乙醇(酸性)置换液5T，氯化钙100Kg，使用盐酸调节乙醇pH为5.5，置换液浓度为76wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 5T of 2.0wt% calcium chloride-ethanol (acidic) replacement solution, 100Kg of calcium chloride, use hydrochloric acid to adjust the pH of ethanol to 5.5, and the concentration of the replacement solution to be 76wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量108KDa)投入到置换液中，开启搅拌进行置换，置换时间为5h。停止搅拌静置约1-2h，抽掉多余上清，加入剩余置换液继续进行二次置换，共计置换2次，每次约5h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 108KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 5h. Stop stirring and let it stand for about 1-2 hours, remove the excess supernatant, add the remaining replacement solution to continue the second replacement, a total of 2 replacements, each time about 5 hours.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为77％、pH为5.93，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 77%, the pH was 5.93, and the washing frequency was 1 time, and the washing time was 3 hours.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为21h，出料得到透明质酸钙18.84kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer 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 21 hours, and the discharge obtains 18.84 kg of calcium hyaluronate.

检测样品指标得出透明质酸钙的钙离子含量为6.7％，钠含量为0.39％，收率为94.18％，分子量为99万，0.1％透明质酸钙溶液的透光率为99.4％，糖醛酸含量48.16％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 6.7%, the sodium content is 0.39%, the yield is 94.18%, the molecular weight is 990,000, the light transmittance of 0.1% calcium hyaluronate solution is 99.4%, sugar The aldehyde acid content is 48.16%.

实施例9Example 9

配制1.0wt％氯化钙-乙醇(酸性)置换液2T，氯化钙20Kg，使用冰乙酸调节乙醇pH为6.5，置换液浓度为75wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.0wt% calcium chloride-ethanol (acidic) replacement solution 2T, calcium chloride 20Kg, use glacial acetic acid to adjust the pH of ethanol to 6.5, and the concentration of the replacement solution to be 75wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量110KDa)投入到置换液中，开启搅拌进行置换，置换时间为5h。停止搅拌静置约1-2h，抽掉多余上清，加入剩余置换液继续进行二次置换，共计置换2次，每次约5h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 110KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 5h. Stop stirring and let it stand for about 1-2 hours, remove the excess supernatant, add the remaining replacement solution to continue the second replacement, a total of 2 replacements, each time about 5 hours.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为72％、pH为5.99，洗涤次数为2次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 72%, the pH was 5.99, and the number of washings was 2 times, and the washing time was 3 hours each time.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为22h，出料得到透明质酸钙18.35kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer 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 22 hours, and 18.35 kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为5.3％，钠含量为0.65％，收率为93.75％，分子量为102万，0.1％透明质酸钙溶液的透光率为99.5％，糖醛酸含量48.18％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 5.3%, the sodium content is 0.65%, the yield is 93.75%, the molecular weight is 1.02 million, the light transmittance of 0.1% calcium hyaluronate solution is 99.5%, sugar The aldehyde acid content is 48.18%.

实施例10Example 10

配制1wt％乙酸钙-乙醇(酸性)置换液10T，乙酸钙10Kg，使用冰乙酸调节乙醇pH为5.2，置换液浓度为80wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 10T of 1wt% calcium acetate-ethanol (acidic) replacement solution, 10Kg of calcium acetate, use glacial acetic acid to adjust the pH of ethanol to 5.2, and the concentration of the replacement solution to be 80wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量30KDa)投入到置换液中，开启搅拌进行置换，置换时间为1h。停止搅拌静置约1-2h，抽掉多余上清，共计置换2次，每次约3h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight: 30KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 1h. Stop stirring and let it stand for about 1-2h, remove excess supernatant, and replace twice in total, about 3h each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为79％、pH为6.07，洗涤次数为2次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 79%, the pH was 6.07, and the number of washings was 2 times, and the washing time was 3 hours each time.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为21h，出料得到透明质酸钙19.78kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer 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 21 hours, and 19.78 kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为3.6％，钠含量为1.8％，收率为96.1％，分子量为4.7万，0.1％透明质酸钙溶液的透光率为99.4％，糖醛酸含量48.79％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 3.6%, the sodium content is 1.8%, the yield is 96.1%, the molecular weight is 47,000, the light transmittance of 0.1% calcium hyaluronate solution is 99.4%, sugar The aldehyde acid content is 48.79%.

实施例11Example 11

配制1wt％氯化钙-乙醇(酸性)置换液10T，氯化钙10Kg，使用冰乙酸调节乙醇pH为5.8，置换液浓度为78wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 10T of 1wt% calcium chloride-ethanol (acidic) replacement solution, 10Kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 5.8, and the concentration of the replacement solution to be 78wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量450KDa)投入到置换液中，开启搅拌进行置换，置换时间为1h。停止搅拌静置约1-2h，抽掉多余上清，共计置换1次，每次约1h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 450KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 1h. Stop stirring and let it stand for about 1-2 hours, remove the excess supernatant, and replace once in total, about 1 hour each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为78％、pH为6.21，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 78%, the pH was 6.21, and the washing frequency was 1 time, and the washing time was 3 hours.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为21h，出料得到透明质酸钙19.67kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 21h, and 19.67kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为3.5％，钠含量为1.4％，收率为98.35％，分子量为3.9万，0.1％透明质酸钙溶液的透光率为99.4％，糖醛酸含量48.34％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 3.5%, the sodium content is 1.4%, the yield is 98.35%, the molecular weight is 39,000, the light transmittance of 0.1% calcium hyaluronate solution is 99.4%, sugar The aldehyde acid content is 48.34%.

实施例12Example 12

配制4wt％氯化钙-乙醇(酸性)置换液1.5T，氯化钙60Kg，使用冰乙酸调节乙醇pH为6.3，置换液浓度为90wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.5T of 4wt% calcium chloride-ethanol (acidic) replacement solution, 60Kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 6.3, and the concentration of the replacement solution is 90wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量8.6KDa)投入到置换液中，开启搅拌进行置换，置换时间为20h。停止搅拌静置约1-2h，抽掉多余上清，共计置换1次，每次约20h。Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight 8.6 KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 20 hours. Stop stirring and let it stand for about 1-2 hours, remove excess supernatant, and replace once in total, about 20 hours each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为78％、pH为6.21，洗涤次数为2次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 78%, the pH was 6.21, and the number of washings was 2 times, and the washing time was 3 hours each time.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为21h，出料得到透明质酸钙19.47kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 21h, and 19.47kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为6.5％，钠含量为0.19％，收率为97.36％，分子量为0.98万，0.1％透明质酸钙溶液的透光率为98.5％，糖醛酸含量48.14％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 6.5%, the sodium content is 0.19%, the yield is 97.36%, the molecular weight is 9,800, and the light transmittance of 0.1% calcium hyaluronate solution is 98.5%. The aldehyde acid content is 48.14%.

对比例1Comparative example 1

配制6wt％氯化钙-乙醇(酸性)置换液1T，氯化钙60Kg，使用冰乙酸调节乙醇pH为5.9，置换液浓度为55wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 6wt% calcium chloride-ethanol (acidic) replacement solution 1T, calcium chloride 60Kg, use glacial acetic acid to adjust the pH of ethanol to 5.9, and the concentration of the replacement solution is 55wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量1570KDa)投入到置换液中，开启搅拌进行置换，置换时间为0.5h。停止搅拌静置约1-2h，抽掉多余上清，共计置换1次，每次约0.5h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 1570KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 0.5h. Stop stirring and let it stand for about 1-2h, remove excess supernatant, replace once in total, about 0.5h each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为50％、pH为4.3，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing liquid equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing liquid was 50%, the pH was 4.3, and the washing frequency was 1 time, and the washing time was 3 hours each time.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为20h，出料得到透明质酸钙19.02kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, and the drying time is 20h, and 19.02kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为3.2％，钠含量为2.8％，收率为95.1％，分子量为138万，0.1％透明质酸钙溶液的透光率为85.7％，糖醛酸含量46.56％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 3.2%, the sodium content is 2.8%, the yield is 95.1%, the molecular weight is 1.38 million, the light transmittance of 0.1% calcium hyaluronate solution is 85.7%, sugar The aldehyde acid content is 46.56%.

对比例2Comparative example 2

配制4wt％氯化钙-乙醇(酸性)置换液1.5T，氯化钙6Kg，使用冰乙酸调节乙醇pH为5.6，置换液浓度为70wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.5T of 4wt% calcium chloride-ethanol (acidic) replacement solution, 6Kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 5.6, and the concentration of the replacement solution is 70wt%. Use a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量1650KDa)投入到置换液中，开启搅拌进行置换，置换时间为24h。停止搅拌静置约1-2h，抽掉多余上清，共计置换2次，每次约24h。Accurately weigh 20kg of sodium hyaluronate raw material (molecular weight 1650KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 24h. Stop stirring and let it stand for about 1-2h, remove excess supernatant, and replace twice in total, about 24h each time.

置换完成后，静置后将上清抽出，加入沉淀体积等倍体积的洗涤液进行洗涤，洗涤液乙醇浓度为75％、pH为6.45，洗涤次数为1次，每次洗涤时间为3h。After the replacement was completed, the supernatant was taken out after standing still, and a washing solution equal to the volume of the precipitate was added for washing. The ethanol concentration of the washing solution was 75%, the pH was 6.45, and the washing frequency was 1 time, and the washing time was 3 hours.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为23h，出料得到透明质酸钙19.13kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 23h, and 19.13kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为2.72％，钠含量为3.2％，收率为95.64％，分子量为115万，0.1％透明质酸钙溶液的透光率为99.1％，糖醛酸含量47.17％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 2.72%, the sodium content is 3.2%, the yield is 95.64%, the molecular weight is 1.15 million, the light transmittance of 0.1% calcium hyaluronate solution is 99.1%, sugar The aldehyde acid content is 47.17%.

对比例3Comparative example 3

配制3wt％氯化钙-乙醇(酸性)置换液1.8T，氯化钙60Kg，使用冰乙酸调节乙醇pH为7.5，置换液浓度为76wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.8T of 3wt% calcium chloride-ethanol (acidic) replacement solution, 60Kg of calcium chloride, use glacial acetic acid to adjust the pH of ethanol to 7.5, and the concentration of the replacement solution is 76wt%. Filter through a 0.45 micron polypropylene filter before use.

准确称取20kg透明质酸钠原料(分子量380KDa)投入到置换液中，开启搅拌进行置换，置换时间为5h。停止搅拌静置约1-2h，抽掉多余上清，共计置换2次，每次约5h。Accurately weigh 20 kg of sodium hyaluronate raw material (molecular weight: 380 KDa) and put it into the replacement solution, start stirring for replacement, and the replacement time is 5 hours. Stop stirring and let it stand for about 1-2 hours, remove excess supernatant, and replace twice in total, about 5 hours each time.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为23h，出料得到透明质酸钙19.46kg。After the washing is completed, the excess supernatant is drawn out, and an ethanol dehydration solution with a volume concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 23h, and 19.46kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为6.9％，钠含量为0.62％，收率为97.3％，分子量为22万，0.1％透明质酸钙溶液的透光率为88.1％，糖醛酸含量48.67％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 6.9%, the sodium content is 0.62%, the yield is 97.3%, the molecular weight is 220,000, the light transmittance of 0.1% calcium hyaluronate solution is 88.1%, sugar The aldehyde acid content is 48.67%.

对比例4Comparative example 4

配制4.0wt％氯化钙-乙醇(酸性)置换液1.5T，氯化钙60kg使用冰乙酸调节乙醇pH为6.2，置换液浓度为45wt％。使用0.45微米聚丙烯滤膜过滤后备用。Prepare 1.5T of 4.0wt% calcium chloride-ethanol (acidic) replacement solution, use glacial acetic acid to adjust the pH of ethanol to 6.2 with 60 kg of calcium chloride, and the concentration of the replacement solution is 45 wt%. Use a 0.45 micron polypropylene filter before use.

洗涤完成后将多余上清抽出，加入沉淀体积等倍体积的浓度为90％的乙醇脱水液进行脱水，脱水时间为0.5～1h，脱水3次。保证上清酒精度在90％以上，然后转入三合一干燥机进行真空干燥，干燥温度为45℃，真空度为0.10MPa，干燥时间为22h，出料得到透明质酸钙16.7kg。After the washing is completed, the excess supernatant is extracted, and an ethanol dehydration solution with a concentration equal to 90% of the precipitation volume is added for dehydration. The dehydration time is 0.5-1 hour, and the dehydration is performed 3 times. Ensure that the alcohol content of the supernatant is above 90%, then transfer to a three-in-one dryer for vacuum drying, the drying temperature is 45°C, the vacuum degree is 0.10MPa, the drying time is 22h, and 16.7kg of calcium hyaluronate is obtained from the discharge.

检测样品指标得出透明质酸钙的钙离子含量为6.5％，钠含量为0.81％，收率为83.5％，分子量为130万，0.1％透明质酸钙溶液的透光率为99.2％，糖醛酸含量47.75％。Detecting the sample indicators shows that the calcium ion content of calcium hyaluronate is 6.5%, the sodium content is 0.81%, the yield is 83.5%, the molecular weight is 1.3 million, the light transmittance of 0.1% calcium hyaluronate solution is 99.2%, sugar The aldehyde acid content is 47.75%.

上述各实施例和对比例的反应条件如表1所示，各实施例和对比例所得到的透明质酸钙的检测结果如表2所示。The reaction conditions of the above-mentioned embodiments and comparative examples are shown in Table 1, and the detection results of calcium hyaluronate obtained in each embodiment and comparative examples are shown in Table 2.

表1实施例和对比例制备方法中的条件Conditions in table 1 embodiment and comparative example preparation method

表2实施例和对比例得到的透明质酸钙的测试结果The test result of the calcium hyaluronate that table 2 embodiment and comparative example obtain

由上述结果可以看出，如实施例2、5与对比例1，4中可以看出，若钙盐浓度太高，为了提高钙盐溶解度，则需降低乙醇的浓度，乙醇浓度过低，则会导致透明质酸钙的收率较低；实施例4与对比例2中可以看出，尽管钙盐浓度不变，置换液酒精度相近的条件下，透明质酸钠和钙盐的投料比中钙盐浓度太低，即使增加置换次数，但同样会导致产品中钙含量偏低，钠含量偏高；实施例6与对比例3中可以看出置换液pH过高会导致钙盐的溶解度降低，产品最终溶液浑浊。As can be seen from the above results, as can be seen in Examples 2 and 5 and Comparative Examples 1 and 4, if the calcium salt concentration is too high, in order to improve the calcium salt solubility, the concentration of ethanol needs to be reduced, and if the concentration of ethanol is too low, then Can cause the yield of calcium hyaluronate to be lower; As can be seen from Example 4 and Comparative Example 2, although the calcium salt concentration is constant, under the condition that the alcohol content of the replacement solution is similar, the feeding ratio of sodium hyaluronate and calcium salt If the concentration of calcium salt in the medium is too low, even if the number of replacements is increased, the calcium content in the product will be low and the sodium content will be high; in Example 6 and Comparative Example 3, it can be seen that the pH of the replacement solution is too high and the solubility of the calcium salt will be reduced. decrease, the final solution of the product is cloudy.

试验例Test case

试验例1透明质酸钙的损伤修复功效Test Example 1 Damage Repair Efficacy of Calcium Hyaluronate

(1)溶液配制。将实施例12中制备的透明质酸钙溶于无血清1640培养液中，0.22μm滤膜过滤除菌，调节浓度至0.025％。(1) Solution preparation. The calcium hyaluronate prepared in Example 12 was dissolved in serum-free 1640 culture solution, sterilized by filtration through a 0.22 μm filter membrane, and the concentration was adjusted to 0.025%.

(2)模拟划痕。将ibidi小室粘贴在24孔板的孔靠近中心位置，使各孔小室方向保持一致。小室的侧壁底部与培养板紧密接触，细胞无法在此处生长，形成模拟划痕。(2) Simulate scratches. Paste the ibidi chamber on the well of the 24-well plate near the center, so that the directions of the chambers in each well are consistent. The bottom of the side wall of the small chamber is in close contact with the culture plate, and cells cannot grow here, forming a simulated scratch.

(3)细胞接种。L929细胞调节密度至1×10 ⁵/mL，向ibidi小室的两个小孔各接种70μL，小室外加入200μL含血清1640培养液，37℃、5％CO ₂条件下培养24h。 (3) Cell inoculation. Adjust the density of L929 cells to 1×10 ⁵ /mL, inoculate 70 μL into each of the two wells of the ibidi chamber, add 200 μL of serum-containing 1640 culture solution outside the chamber, and culture at 37°C and 5% CO ₂ for 24 hours.

(4)给药。移除ibidi小室，弃培养液，加入透明质酸钙的无血清培养液。(4) Administration. Remove the ibidi chamber, discard the culture medium, and add calcium hyaluronate serum-free culture medium.

(5)数据处理。继续培养24h，用Image J软件计算修复面积。(5) Data processing. Continue to cultivate for 24 hours, and calculate the repair area with Image J software.

细胞迁移率(％)＝(1-加药后划痕区域面积/初始划痕区域面积)×100Cell mobility (%)=(1-area of scratched area after drug addition/area of initial scratched area)×100

结果和分析：Results and Analysis:

结果表3所示，与模型组对比，透明质酸钙在0.025％浓度下，表现出促进细胞迁移和创伤愈合的作用。其中，愈合率等于细胞迁移率。The results are shown in Table 3. Compared with the model group, calcium hyaluronate exhibited the effect of promoting cell migration and wound healing at a concentration of 0.025%. where the healing rate is equal to the cell migration rate.

表3透明质酸钙的损伤修复的影响Table 3 Effect of Calcium Hyaluronate on Damage Repair

其中，T0划痕面积表示初始划痕面积，T24h划痕面积表示使用透明质酸钙24h后的数值。Among them, the T0 scratch area represents the initial scratch area, and the T24h scratch area represents the value after using calcium hyaluronate for 24 hours.

试验例2透明质酸钙人体皮肤功效评价Test Example 2 Calcium Hyaluronate Human Skin Efficacy Evaluation

招募健康受试者10名，用半脸对照的方法进行测试，Recruit 10 healthy subjects, and use half-face control method to test,

左脸使用含实施例12制备的，浓度0.5％透明质酸钙的乳液，右脸使用空白乳液。Use the emulsion containing 0.5% calcium hyaluronate prepared in Example 12 on the left face, and use blank emulsion on the right face.

于使用透明质酸钙产品前后分别检测受试者面部苹果肌区域的角质层含水量、经皮水分散失量、纹理面积。Before and after using calcium hyaluronate products, the water content of the stratum corneum, the amount of transdermal water loss, and the texture area of the apple muscle area of the subject's face were detected.

受试者洁面后30分钟，检测左、右半脸苹果肌区域的角质层含水量、经皮水分散失量、纹理面积的初始值，然后分别使用等量的透明质酸钙乳液和空白乳液，早晚各使用一次，并于使用后1W、2W、4W再次检测所有指标。30 minutes after the subject cleansed his face, the initial values of the stratum corneum water content, transepidermal water loss, and texture area in the apple muscle area of the left and right half of the face were measured, and then the same amount of calcium hyaluronate emulsion and blank emulsion were used respectively. Use it once in the morning and evening, and check all indicators again at 1W, 2W, and 4W after use.

其中，角质层含水量使用皮肤水分测试仪Corneometer CM 825(Courage+Khazaka，德国)进行检测。Among them, the water content of the stratum corneum was detected by a skin moisture tester Corneometer CM 825 (Courage+Khazaka, Germany).

经皮水分散失量使用皮肤水分散失测定仪

TM Hex(Courage+Khazaka，德国)进行检测。 Transdermal water loss using a skin water loss meter

TM Hex (Courage+Khazaka, Germany) for detection.

纹理面积使用VISIA CR面部图像分析***(Canfield，美国)进行检测。The texture area was detected using the VISIA CR facial image analysis system (Canfield, USA).

结果如表4-表6所示。The results are shown in Table 4-Table 6.

表4透明质酸钙对角质层含水量的影响Table 4 Effect of Calcium Hyaluronate on Water Content of Cuticle

the	T0(％)T0(%)	T1W(％)T1W(%)	T2W(％)T2W(%)	T4W(％)T4W(%)
透明质酸钙calcium hyaluronate	100.00100.00	132.55132.55	145.47145.47	133.83133.83
空白blank	100.00100.00	131.41131.41	135.62135.62	128.60128.60

其中，T0表示初始含水量，以100％表示，T1W表示使用透明质酸钙1周后角质层含水量与初始含水量的比值，T2W表示使用透明质酸钙2周后角质层含水量与初始含水量的比值，T4W表示使用透明质酸钙4周后角质层含水量与初始含水量的比值。Among them, T0 represents the initial water content, expressed as 100%, T1W represents the ratio of the water content of the stratum corneum to the initial water content after using calcium hyaluronate for 1 week, and T2W represents the ratio of the water content of the stratum corneum to the initial water content after using calcium hyaluronate for 2 weeks The ratio of water content, T4W represents the ratio of the water content of the stratum corneum to the initial water content after using calcium hyaluronate for 4 weeks.

结果显示，与空白组相比，透明质酸钙在使用后1周-4周，角质层含水量较高(提高5％-10％)，说明透明质酸钙可提高皮肤角质层含水量。The results showed that, compared with the blank group, the water content of the stratum corneum was higher (increased by 5%-10%) after 1 week to 4 weeks of use of calcium hyaluronate, indicating that calcium hyaluronate can increase the water content of the stratum corneum of the skin.

表5透明质酸钙对经皮水分散失量的影响Table 5 Effect of calcium hyaluronate on transdermal water loss

the	T0(％)T0(%)	T1W(％)T1W(%)	T2W(％)T2W(%)	T4W(％)T4W(%)
透明质酸钙calcium hyaluronate	100.00100.00	81.0581.05	93.2793.27	98.1398.13
空白blank	100.00100.00	92.3792.37	98.4598.45	98.9898.98

结果显示，与空白组相比，透明质酸钙在使用后2周内经皮水分散失量明显降低，特别是第1周时降低11％.说明透明质酸钙可减少经皮水分散失量。The results showed that, compared with the blank group, the transdermal water loss of calcium hyaluronate was significantly reduced within 2 weeks after use, especially 11% in the first week. It shows that calcium hyaluronate can reduce the transdermal water loss.

表6透明质酸钙对皮肤细纹的影响Table 6 Effect of Calcium Hyaluronate on Skin Fine Lines

the	T0(％)T0(%)	T1W(％)T1W(%)	T2W(％)T2W(%)	T4W(％)T4W(%)
透明质酸钙calcium hyaluronate	100.00100.00	111.52111.52	112.89112.89	112.15112.15
空白blank	100.00100.00	111.56111.56	114.31114.31	118.66118.66

其中，T0表示初始纹理面积，以100％表示，T1W表示使用透明质酸钙1周后纹理面积与初始纹理面积的比值，T2W表示使用透明质酸钙2周后纹理面积与初始纹理面积的比值，T4W表示使用透明质酸钙4周后纹理面积与初始纹理面积的比值。Among them, T0 represents the initial texture area, expressed as 100%, T1W represents the ratio of the texture area to the initial texture area after using calcium hyaluronate for 1 week, and T2W represents the ratio of the texture area to the initial texture area after using calcium hyaluronate for 2 weeks , T4W represents the ratio of texture area to initial texture area after using calcium hyaluronate for 4 weeks.

结果显示，透明质酸钙在使用2周后开始发挥减少皮肤细纹的功效，至使用4周时与空白组间差异显著(p<0.05)，比空白组纹理面积减少6％。说明透明质酸钙有减少皮肤细纹的功效。The results showed that calcium hyaluronate began to reduce skin fine lines after 2 weeks of use, and there was a significant difference (p<0.05) between the 4 weeks of use and the blank group, and the texture area was reduced by 6% compared with the blank group. It shows that calcium hyaluronate has the effect of reducing fine lines on the skin.

试验例3抗氧化实验Test Example 3 Antioxidation Experiment

1、清除羟自由基实验1. Hydroxyl free radical scavenging experiment

(1)实验方法：(1) Experimental method:

溶液配制：Solution preparation:

8.8mM的过氧化氢：精确称取0.0997g，用纯化水定溶于到100mL棕色容量瓶。8.8mM hydrogen peroxide: Accurately weigh 0.0997g, and dissolve it in purified water into a 100mL brown volumetric flask.

9mM的硫酸亚铁：精确称取0.2502g，用纯化水定溶于到100mL棕色容量瓶。9mM ferrous sulfate: Accurately weigh 0.2502g, and dissolve it in purified water into a 100mL brown volumetric flask.

9mM的水杨酸-乙醇：精确称取0.1243g，用无水乙醇定溶于到100mL棕色容量瓶。9mM salicylic acid-ethanol: Accurately weigh 0.1243g, and dissolve it in absolute ethanol into a 100mL brown volumetric flask.

(2)实验样品：(2) Experimental samples:

样品溶液的制备方法为：将表7中的相应成分溶解于纯化水中，配成相应浓度的溶液。The preparation method of the sample solution is as follows: the corresponding components in Table 7 are dissolved in purified water to prepare solutions of corresponding concentrations.

表7Table 7

(3)实验过程：(3) Experimental process:

取试管若干只，首先用移液管加入9mM硫酸亚铁0.2mL，9mM水杨酸-乙醇溶液0.2mL，样品溶液3.0mL，最后加8.8mM的过氧化氢溶液0.25mL启动反应。37℃反应30分钟，以纯化水作为参比，在510nm处测定光吸收值。考虑到样品本身的吸光值，以硫酸亚铁、水杨酸-乙醇溶液、样品溶液和蒸馏水作为样品的本底吸收值。每个样品做两个对照，本底吸收值做两个对照。按下式计算·OH清除率。Take several test tubes, first add 0.2mL of 9mM ferrous sulfate, 0.2mL of 9mM salicylic acid-ethanol solution, 3.0mL of sample solution with a pipette, and finally add 0.25mL of 8.8mM hydrogen peroxide solution to start the reaction. React at 37°C for 30 minutes, and measure the light absorption value at 510 nm with purified water as a reference. Considering the absorbance value of the sample itself, ferrous sulfate, salicylic acid-ethanol solution, sample solution and distilled water were used as the background absorbance value of the sample. Make two controls for each sample, and make two controls for the background absorbance. Calculate the OH scavenging rate according to the formula.

(4)结果计算：(4) Result calculation:

其中，A ₀为空白对照液的吸光度，Ax为加入样品溶液后的吸光度，A _x0为不加显色剂过氧化氢样品溶液本底的吸光度。 Among them, A ₀ is the absorbance of the blank control solution, Ax is the absorbance after adding the sample solution, and _Ax0 is the absorbance of the background of the sample solution without adding the chromogenic agent hydrogen peroxide.

(5)实验结果(5) Experimental results

不同样品的·OH自由基清除情况如图1和图2所示。The OH radical scavenging conditions of different samples are shown in Fig. 1 and Fig. 2.

从图1可以看出，相同浓度条件下，不同分子量的透明质酸钙都表现出了明显的清除羟自由基的能力，且不同分子量的透明质酸钙对羟自由基的清除能力不同，分子量越低的透明质酸钙羟自由基清除能力越强；另外，D1、D2和D4说明，单独的氯化钙和透明质酸钠几乎没有清除羟自由基的能力。而通过S3和D1以及S17和D4的对比可知，透明质酸钙的效果均要高于相同分子量下透明质酸钠的效果，这说明透明质酸钙在清除羟自由基方面与透明质酸钠相比也具有明显的增效作用。D3说明，透明质酸钠和氯化钙混合物具有一定的清除羟自由基的能力，但效果低于同分子量段的透明质酸钙。从图2可以看出:相同分子量条件下，不同浓度的透明质酸钙也都表现出了明显的清除羟自由基的能力，且浓度越高透明质酸钙清除羟自由基的能力越强。It can be seen from Figure 1 that under the same concentration conditions, calcium hyaluronate with different molecular weights all showed obvious ability to scavenge hydroxyl free radicals, and calcium hyaluronate with different molecular weights had different scavenging abilities to hydroxyl free radicals. The lower the calcium hyaluronate, the stronger the hydroxyl radical scavenging ability; in addition, D1, D2 and D4 indicated that calcium chloride and sodium hyaluronate alone had almost no ability to scavenge hydroxyl radicals. And through the comparison of S3 and D1 and S17 and D4, it can be seen that the effect of calcium hyaluronate is higher than that of sodium hyaluronate at the same molecular weight, which shows that calcium hyaluronate is comparable to sodium hyaluronate in scavenging hydroxyl radicals. It also has obvious synergistic effect. D3 shows that the mixture of sodium hyaluronate and calcium chloride has a certain ability to scavenge hydroxyl radicals, but the effect is lower than that of calcium hyaluronate with the same molecular weight. As can be seen from Fig. 2: under the same molecular weight condition, the calcium hyaluronate of different concentrations also all showed the obvious ability of scavenging hydroxyl free radicals, and the higher the concentration, the stronger the ability of calcium hyaluronate to scavenge hydroxyl free radicals.

2、清除超氧阴离子自由基的实验2. Experiments on scavenging superoxide anion free radicals

(1)实验方法：(1) Experimental method:

溶液配制：50mmolL-1 Tris-HCL缓冲液(pH8.2)：称取1.5140g Tris固体用适量纯化水溶于250mL三角瓶中，溶解后加入0.4771mL浓盐酸，加入0.0514gEDTA后定容到250mL，装入棕色瓶备用，测定pH为8.2。Solution preparation: 50mmolL-1 Tris-HCL buffer solution (pH8.2): Weigh 1.5140g Tris solid and dissolve it in a 250mL Erlenmeyer flask with an appropriate amount of purified water. After dissolving, add 0.4771mL concentrated hydrochloric acid, add 0.0514gEDTA, and then dilute to 250mL. Put it into a brown bottle for later use, and measure the pH to be 8.2.

50mM邻苯三酚：称取0.0630g邻苯三酚，溶于10mM Hcl(吸取0.0833mL浓盐酸稀释到100mL)装入棕色瓶备用。50mM pyrogallol: Weigh 0.0630g of pyrogallol, dissolve in 10mM Hcl (dilute to 100mL by absorbing 0.0833mL of concentrated hydrochloric acid) and put it into a brown bottle for later use.

8mM的盐酸：用移液器精确吸取66.7ul用纯化水定容到100mL。8mM hydrochloric acid: Use a pipette to accurately draw 66.7ul and dilute to 100mL with purified water.

(2)实验样品：(2) Experimental samples:

实验样品与清除羟自由基实验一致。The experimental sample is consistent with the scavenging hydroxyl radical experiment.

(3)实验过程(3) Experimental process

取离心管若干只，用移液管加入50mmolL-1 Tris-Hcl缓冲液(pH8.2)3.0mI，置25℃水浴中保温20min，分别加人3.0mL样品溶液和0.1mL50mmolL-1邻苯三酚溶液，混匀后于25℃水浴中反应5min，加入1.0mL 8mM Hcl终止反应，于320nm处测定吸光度(Ax)，空白对照组以相同体积蒸馏水代替样品(A0)。每个试样做2个平行，取平均值，按下式计算清除率。Take several centrifuge tubes, add 50mmolL-1 Tris-Hcl buffer solution (pH8.2) 3.0mI with a pipette, put it in a water bath at 25°C for 20min, add 3.0mL sample solution and 0.1mL 50mmolL-1 phthalate tris The phenol solution was mixed and reacted in a water bath at 25°C for 5 minutes, and 1.0mL of 8mM Hcl was added to terminate the reaction, and the absorbance (Ax) was measured at 320nm. The blank control group was replaced with the same volume of distilled water as the sample (A0). Make 2 parallels for each sample, take the average value, and calculate the clearance rate according to the following formula.

(4)结果计算(4) Calculation of results

其中，Ax为样品的吸光度值，A ₀为空白对照的吸光度值，参比溶液为Tris-Hcl缓冲液。 Among them, Ax is the absorbance value of the sample, _A0 is the absorbance value of the blank control, and the reference solution is Tris-Hcl buffer.

(5)实验结果(5) Experimental results

不同样品的超氧阴离子自由基清除情况如图3和图4所示。The superoxide anion radical scavenging conditions of different samples are shown in Figure 3 and Figure 4.

从图3可以看出：相同浓度条件下，不同分子量的透明质酸钙都表现出了明显的清除超氧离子自由基的能力，且不同分子量的透明质酸钙对超氧离子自由基的清除能力不同，分子量越低的透明质酸钙超氧离子自由基清除能力越强；另外，D1、D2和D4说明，单独的氯化钙和透明质酸钠几乎没有清除超氧阴离子自由基的能力。D3说明，透明质酸钠和氯化钙混合物具有一定的清除超氧阴离子自由基的能力，但效果低于同分子量段的透明质酸钙，这说明透明质酸钙在清除超氧离子自由基方面与透明质酸钠和氯化钙的混合物相比也具有明显的增效作用。从图4可以看出:相同分子量条件下，不同浓度的透明质酸钙也都表现出了明显的清除超氧离子的能力，且浓度越高透明质酸钙清除超氧离子的能力越强。It can be seen from Figure 3 that: under the same concentration conditions, calcium hyaluronate with different molecular weights has shown obvious ability to scavenge superoxide ion free radicals, and calcium hyaluronate with different molecular weights has the ability to scavenge superoxide ion free radicals. Different abilities, the lower the molecular weight of calcium hyaluronate, the stronger the ability to scavenge superoxide anion radicals; in addition, D1, D2 and D4 show that independent calcium chloride and sodium hyaluronate have almost no ability to scavenge superoxide anion radicals . D3 shows that the mixture of sodium hyaluronate and calcium chloride has a certain ability to scavenge superoxide anion free radicals, but the effect is lower than that of calcium hyaluronate with the same molecular weight, which shows that calcium hyaluronate has the ability to scavenge superoxide anion free radicals. Compared with the mixture of sodium hyaluronate and calcium chloride, it also has obvious synergistic effect. As can be seen from Fig. 4: under the same molecular weight condition, the calcium hyaluronate of different concentrations has also all shown the obvious ability of removing superoxide ion, and the higher concentration is stronger the ability of removing superoxide ion of calcium hyaluronate.

综合以上实验结果可知，透明质酸钙具有显著的羟自由基、超氧阴离子自由基的清除能力，因此显示透明质酸钙具有良好的抗氧化能力，可广泛应用于具有抗氧化用途的医药、化妆品、医美等产品中。Based on the above experimental results, it can be seen that calcium hyaluronate has a significant ability to scavenge hydroxyl radicals and superoxide anion radicals, so it shows that calcium hyaluronate has good antioxidant capacity and can be widely used in medicines, In cosmetics, medical beauty and other products.

Claims

一种透明质酸钙的制备方法，其特征在于，包括以下步骤：A preparation method of calcium hyaluronate, is characterized in that, comprises the following steps:

将透明质酸钠加入到含有钙盐的酸性置换液中，进行置换，得到透明质酸钙沉淀；adding sodium hyaluronate to the acidic replacement solution containing calcium salts for replacement to obtain calcium hyaluronate precipitates;

用酸性洗涤液洗涤所述沉淀；Washing the precipitate with an acid washing solution;

将洗涤后的所述沉淀用中性脱水液进行脱水，真空干燥，得到透明质酸钙粉末；Dehydrating the washed precipitate with a neutral dehydrating solution and drying it in vacuum to obtain calcium hyaluronate powder;

其中所述置换液中有机介质浓度为50％～90wt％，优选为60％～80wt％，钙盐浓度为0.5％～4wt％，优选为1％～3wt％。Wherein the replacement liquid has an organic medium concentration of 50%-90wt%, preferably 60%-80wt%, and a calcium salt concentration of 0.5%-4wt%, preferably 1%-3wt%.
根据权利要求1所述的制备方法，其特征在于，所述有机介质为与水相容性好、但透明质酸钠或透明质酸钙在其中不溶或微溶的有机介质，优选为醇类有机溶剂或酮类有机溶剂，更优选为乙醇、甲醇或丙酮。The preparation method according to claim 1, wherein the organic medium is an organic medium with good water compatibility but insoluble or slightly soluble in sodium hyaluronate or calcium hyaluronate, preferably alcohols An organic solvent or a ketone organic solvent, more preferably ethanol, methanol or acetone.
根据权利要求1所述的制备方法，其特征在于，透明质酸钠的分子量为3kDa～2200kDa。The preparation method according to claim 1, characterized in that the molecular weight of sodium hyaluronate is 3kDa～2200kDa.
根据权利要求1所述的制备方法，其特征在于，透明质酸钠与钙盐的质量比为1:0.5～5，优选为1:1～3。The preparation method according to claim 1, characterized in that the mass ratio of sodium hyaluronate to calcium salt is 1:0.5-5, preferably 1:1-3.
根据权利要求1所述的制备方法，其特征在于，所述置换液和所述洗涤液的pH分别为5.0～7.0，所述洗涤液中有机介质浓度为50％～90wt％，优选为60％～80wt％，所述脱水液中有机介质浓度大于等于90wt％。The preparation method according to claim 1, characterized in that, the pH of the replacement liquid and the washing liquid are 5.0-7.0 respectively, and the concentration of the organic medium in the washing liquid is 50%-90wt%, preferably 60% ~80wt%, the concentration of the organic medium in the dehydration liquid is greater than or equal to 90wt%.
根据权利要求1所述的制备方法，其特征在于，置换的次数为1～4次，优选为1～2次，置换时间为1～24h，优选为5～16h；优选地，洗涤次数为1～5次，优选为2～3次；进一步优选地，脱水次数为1～5次，优选为2～3次。The preparation method according to claim 1, characterized in that, the number of replacements is 1 to 4 times, preferably 1 to 2 times, and the replacement time is 1 to 24 hours, preferably 5 to 16 hours; preferably, the number of times of washing is 1 ~5 times, preferably 2~3 times; more preferably, the number of dehydration is 1~5 times, preferably 2~3 times.
根据权利要求1所述的制备方法，其特征在于，所述钙盐选自氯化钙、硝酸钙、乙酸钙、甲酸钙、亚硝酸钙中的一种或两种以上。The preparation method according to claim 1, wherein the calcium salt is selected from one or more of calcium chloride, calcium nitrate, calcium acetate, calcium formate, and calcium nitrite.
根据权利要求1所述的制备方法，其特征在于，所得到的透明质酸钙粉末中透明质酸钙的含量大于等于90wt％，在1wt％水溶液中的透光率大于等于99％。The preparation method according to claim 1, characterized in that the calcium hyaluronate content in the obtained calcium hyaluronate powder is greater than or equal to 90 wt%, and the light transmittance in a 1 wt% aqueous solution is greater than or equal to 99%.
一种透明质酸钙，其特征在于，所述透明质酸钙的分子量为2kDa-2000kDa，优选为2kDa-1000kDa。A calcium hyaluronate, characterized in that the molecular weight of the calcium hyaluronate is 2kDa-2000kDa, preferably 2kDa-1000kDa.
根据权利要求9所述的透明质酸钙，其特征在于，在1wt％水溶液中所述透明质酸钙的透光率大于等于99％。The calcium hyaluronate according to claim 9, characterized in that the light transmittance of the calcium hyaluronate in a 1wt% aqueous solution is greater than or equal to 99%.
根据权利要求9或10所述的透明质酸钙，其特征在于，所述透明质酸钙通过将透明质酸钠加入到含有钙盐的酸性置换液中进行置换得到，所述置换液中有机介质浓度为60％～80wt％，钙盐浓度为1％～3wt％，所述置换液pH为5.0～7.0。The calcium hyaluronate according to claim 9 or 10, characterized in that, the calcium hyaluronate is obtained by adding sodium hyaluronate into an acidic replacement solution containing calcium salts for replacement, and the replacement solution contains organic The medium concentration is 60%-80wt%, the calcium salt concentration is 1%-3wt%, and the pH of the replacement solution is 5.0-7.0.
权利要求1-8中任一项所述的制备方法制备得到的透明质酸钙，或权利要求9-11中任一项所述的透明质酸钙，在损伤修复、提高皮肤角质层含水量、减少皮肤细纹、抗氧化中的应用。The calcium hyaluronate prepared by the preparation method described in any one of claims 1-8, or the calcium hyaluronate described in any one of claims 9-11, is effective in repairing damage and increasing the water content of the stratum corneum of the skin , reduce skin fine lines, anti-oxidation in the application.
一种损伤修复、提高皮肤角质层含水量、减少皮肤细纹、抗氧化的方法，其包括向受试者施用权利要求1-8中任一项所述的制备方法制备得到的透明质酸钙，或权利要求9-11中任一项所述的透明质酸钙。A method for repairing damage, increasing the water content of the stratum corneum of the skin, reducing fine lines of the skin, and anti-oxidation, comprising administering to a subject the calcium hyaluronate prepared by the preparation method described in any one of claims 1-8 , or the calcium hyaluronate described in any one of claims 9-11.
一种组合物，其包括权利要求9-11中任一项所述的透明质酸钙。A composition comprising the calcium hyaluronate according to any one of claims 9-11.