Sodium hyaluronate having full molecular weight distribution, preparation method therefor, and use thereof

Abstract

The present invention provides a preparation method for sodium hyaluronate having full molecular weight distribution, the method comprising: step 1): spraying hydrogen peroxide and performing ultraviolet irradiation on a sodium hyaluronate solid raw material, and obtaining a sodium hyaluronate degraded material; step 2): dissolving the sodium hyaluronate degraded material in water, using an NaOH solution to adjust the pH to alkaline, and obtaining a sodium hyaluronate alkaline solution; step 3): ultrasonically treating the sodium hyaluronate alkaline solution; step 4): preparing hyaluronic acid solid raw material into a sodium hyaluronate solution having a concentration of 0.1%-1% (w / v), and mixing evenly with the ultrasonically treated sodium hyaluronate alkaline solution at an addition ratio of 20%-60% (v / v); and step 5): performing adsorption treatment by using diatomite and activated carbon, respectively, filtering and concentrating by using a nanofiltration membrane, and then drying to obtain the sodium hyaluronate having full molecular weight distribution. The preparation method of the present invention can obtain sodium hyaluronate having good water-replenishing and moisturizing, anti-inflammatory, cell protection, and other activity.

Description

Sodium hyaluronate with full molecular weight distribution and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application CN 202110874694.1, filed on July 30, 2021, entitled “A Sodium Hyaluronate with Full Molecular Weight Distribution, Preparation Method and Application Thereof”, and the entire contents of the above application are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biomaterial technology, and in particular to a sodium hyaluronate with a full molecular weight distribution, a preparation method thereof, and an application thereof. Background Art

[0004] Hyaluronic acid (HA) is a linear polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine. Its molecular weight ranges from hundreds of thousands to millions of daltons. It is widely distributed in animal tissues, with highest concentrations found in human skin, umbilical cord, vitreous humor, and synovial fluid. It is also widely distributed in plants and microorganisms. In nature, HA has a consistent structure, exhibits no racial differences, and is non-immunogenic.

[0005] When HA is at a high concentration (1%), the molecules exist in a network form and have high viscoelasticity. The aqueous solution of HA is a viscoelastic fluid that fills the space between cells and collagen fibers and covers certain epidermal tissues. Its main function is to protect and lubricate cells and regulate the movement of cells on this elastic matrix. In the skin, HA can lock in moisture, make the skin smooth and tender, and maintain skin elasticity. HA in the synovial fluid of human joints can lubricate joints, isolate the stimulation of harmful signal factors, and protect joint cartilage. HA in the vitreous body of the eye plays the function of supporting the eye chamber, protecting the retina, and making the eyes bright. HA is internationally recognized as an ideal natural moisturizing factor and can be used in various cosmetics to achieve the effect of beauty and moisturizing. After oral absorption, HA can participate in the synthesis of HA in the body and is distributed in human skin, joint cavity and vitreous body of the eye. It can supplement hyaluronic acid in the synovial fluid of the joints and increase skin elasticity.

[0006] HA is mostly produced and used in the form of sodium hyaluronate. The efficacy of different types of sodium hyaluronate varies greatly. For example, high molecular weight sodium hyaluronate (molecular weight > 500,000) has a better mucosal protective effect, while low molecular weight sodium hyaluronate (molecular weight 10,000-500,000) and oligomeric sodium hyaluronate (molecular weight <10,000) have better advantages in skin penetration and oral absorption. At present, hyaluronic acid products are mainly concentrated in hyaluronic acid with a single molecular weight distribution, which has a narrow molecular weight distribution width and uneven quality. Therefore, in order to obtain sodium hyaluronate with a wider molecular weight distribution, the existing methods need to mix and compound sodium hyaluronate of each molecular weight range. However, the viscosity and other properties of sodium hyaluronate products with different molecular weights vary greatly, and the uniformity of the product obtained by direct mixing is difficult to ensure. The existing technology also provides some physical, chemical or biological methods for the degradation of sodium hyaluronate. However, the molecular weight distribution of sodium hyaluronate treated by existing degradation methods is not uniform, and the stability is also poor, making it difficult to further improve the quality of sodium hyaluronate products.

[0007] Summary of the Invention

[0008] In order to solve the above problems, the present invention aims to provide a high-quality sodium hyaluronate with good uniformity, high stability and full molecular weight distribution, as well as a preparation method and application thereof.

[0009] In one aspect, the present application provides a method for preparing sodium hyaluronate with a full molecular weight distribution, the method comprising:

[0010] Step 1): spraying the sodium hyaluronate solid raw material with hydrogen peroxide and irradiating it with ultraviolet light to obtain a sodium hyaluronate degradation material;

[0011] Step 2): dissolving the sodium hyaluronate degradation material in water and adjusting the pH to alkaline with a NaOH solution to obtain a sodium hyaluronate alkaline solution;

[0012] Step 3): ultrasonically treating the sodium hyaluronate alkaline solution;

[0013] Step 4): The sodium hyaluronate solid raw material is prepared into a sodium hyaluronate solution with a concentration of 0.1% to 1% (w / v), and the sodium hyaluronate alkaline solution obtained in step 3) after ultrasonication is mixed uniformly at a ratio of 20% to 60% (v / v);

[0014] Step 5): Adsorption treatment is performed using diatomaceous earth and activated carbon respectively, and filtration and concentration are performed using a nanofiltration membrane. After drying, the sodium hyaluronate with a full molecular weight distribution is obtained.

[0015] Furthermore, in the step 1), 50 to 100 ml of hydrogen peroxide with a mass concentration of 1% to 5% is sprayed per kilogram of the sodium hyaluronate solid raw material, and the ultraviolet irradiation dose is 300 to 1500 μW / cm 2 The irradiation time is 50 to 70 minutes.

[0016] It is understood that the UV irradiation dose in this application refers to the UV light flux received per unit area. Optionally, the UV irradiation step is to place the device under a UV lamp for irradiation, wherein the UV lamp has an irradiation wavelength of 200-400nm, preferably 220-320nm.

[0017] Furthermore, the molecular weight of the sodium hyaluronate solid raw material is 1 to 2 million.

[0018] It is understood that the molecular weight of sodium hyaluronate described in this application refers to its weight average molecular weight.

[0019] Furthermore, the solid raw material of sodium hyaluronate used is selected from one or more of cosmetic grade sodium hyaluronate, food grade sodium hyaluronate, and pharmaceutical grade sodium hyaluronate.

[0020] Furthermore, in the step 2), the volume concentration of the sodium hyaluronate degradation material after being dissolved in water is 1% to 10% (w / v); and / or,

[0021] Adjust the pH to 10-12 with NaOH solution.

[0022] Furthermore, in the step 3), the frequency of the ultrasonic treatment is 10 to 100 kHz, and the time is 15 to 180 minutes.

[0023] Furthermore, in the step 5),

[0024] The step of using diatomaceous earth adsorption treatment includes: adding 0.1% to 1% (w / v) diatomaceous earth based on the mass of the sodium hyaluronate alkaline solution, stirring and adsorbing at 45-80° C. for 30 to 60 minutes, and filtering; and / or,

[0025] The steps of using activated carbon adsorption treatment include: adjusting the pH to 6-7 with a dilute acid solution, adding 0.1%-1% (w / v) activated carbon based on the mass of the sodium hyaluronate solution, stirring and adsorbing at 45-80° C. for 30-60 minutes, and filtering; preferably, the dilute acid solution is selected from one or more of a dilute hydrochloric acid solution, a dilute sulfuric acid solution, a dilute acetic acid solution, and a dilute hypochlorous acid solution.

[0026] Furthermore, in step 5), the molecular weight cut-off of the nanofiltration membrane is 200-300 Da, the operating pressure is controlled at 15-30 bar, and the temperature is controlled at 30-50°C.

[0027] On the other hand, the present application also provides sodium hyaluronate with a full molecular weight distribution prepared by the above method, wherein the weight average molecular weight of the sodium hyaluronate with a full molecular weight distribution ranges from 2,000 to 1.5 million, and the molecular weight dispersion coefficient Mw / Mn is greater than 5;

[0028] Preferably, the endotoxin content in the sodium hyaluronate is <0.01EU, and the protein content is <0.01%;

[0029] Preferably, the OD value of the sodium hyaluronate at 280 nm is <0.01, and the OD value at 260 nm is <0.01.

[0030] On the other hand, the present application also provides the use of sodium hyaluronate prepared by the above-mentioned preparation method in the preparation of moisturizers, lubricants, anti-inflammatory agents and / or cell repair agents for medical, food and / or cosmetic use.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The preparation method provided by the present invention can quickly and efficiently prepare sodium hyaluronate with a full molecular weight distribution, and the prepared sodium hyaluronate has good uniformity and high stability, and can fully utilize the efficacy advantages of sodium hyaluronate of various molecular weights, with a molecular weight dispersion coefficient Mw / Mn of above 5.

[0033] 2. The preparation method of full molecular weight sodium hyaluronate provided by the present invention combines ultraviolet irradiation degradation, ultrasound-alkaline hydrolysis treatment, diatomaceous earth filtration, activated carbon adsorption and nanomembrane filtration. The obtained sodium hyaluronate has an endotoxin content of <0.01EU, a protein content of <0.01%, an OD value at 280nm of <0.01, and an OD value at 260nm of <0.01. It has higher biocompatibility and can achieve a higher quality level, which is beneficial to improving the application effect of sodium hyaluronate in skin moisturizing, cell protection, oral administration, etc.

[0034] 3. The preparation method of full molecular weight sodium hyaluronate provided by the present invention places ultraviolet irradiation degradation and ultrasonic degradation before filtration of high-viscosity hyaluronic acid solution, which greatly reduces the difficulty of filtering high-viscosity solution, improves preparation efficiency and yield, and improves impurity removal efficiency.

[0035] 4. The preparation method of full molecular weight sodium hyaluronate provided by the present invention obtains high-quality full molecular weight distribution sodium hyaluronate with excellent intelligent hydrating, moisturizing, anti-inflammatory and cell protection activities. DETAILED DESCRIPTION

[0036] In order to more clearly illustrate the overall concept of the present application, the following examples are described in detail. It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention.

[0037] Example 1:

[0038] This embodiment provides a method for preparing sodium hyaluronate with a full molecular weight distribution, which specifically comprises the following steps:

[0039] Step 1): Take 1 kg of food-grade sodium hyaluronate raw material with a molecular weight of 1 million, and evenly spray 50 mL of 1% hydrogen peroxide on the surface of the sodium hyaluronate raw material at 300 μW / cm 2 The dose was UV irradiated for 60 min;

[0040] Step 2): Dissolve the UV-irradiated sodium hyaluronate in water at a concentration of 1% (w / v) and stir at room temperature until it is completely dissolved;

[0041] Step 3): The pH was adjusted to 10 using a 2% NaOH solution, and then the mixture was subjected to ultrasonic treatment at 10 kHz for 15 min.

[0042] Step 4): Prepare 1 million sodium hyaluronate solution with a concentration of 1% (w / v), and add 60% (v / v) to mix with the above-treated sodium hyaluronate alkaline solution;

[0043] Step 5): using 0.1% diatomaceous earth, stirring at 45° C., adsorption treatment for 30 min, and filtering;

[0044] Step 6): After adjusting the pH to 5.0 with 2% dilute hydrochloric acid solution, adsorb with 0.1% activated carbon at 45°C with stirring for 30 minutes, and filter;

[0045] Step 7): The filtrate is concentrated using a nanofiltration membrane system, wherein the nanofiltration membrane has a molecular weight cutoff of 300 Da, an operating pressure of 15 bar, and a temperature controlled at 40° C. to obtain a nanofiltration concentrate, which is spray-dried to obtain a high-quality sodium hyaluronate sample with a full molecular weight distribution.

[0046] Example 2:

[0047] This embodiment provides a method for preparing sodium hyaluronate with a full molecular weight distribution, which specifically comprises the following steps:

[0048] Step 1): Take 1 kg of food-grade sodium hyaluronate raw material with a molecular weight of 1.2 million, and evenly spray 80 mL of 3% hydrogen peroxide on the surface of the sodium hyaluronate material. 2 The dose was UV irradiated for 60 min;

[0049] Step 2): Dissolve the UV-irradiated sodium hyaluronate in water at a concentration of 3% (w / v) and stir at room temperature until it is completely dissolved;

[0050] Step 3): The pH was adjusted to 12 using a 5% NaOH solution, and then the mixture was subjected to ultrasound at 30 kHz for 30 min.

[0051] Step 4): Prepare 1.2 million sodium hyaluronate raw material solution with a concentration of 0.8% (v / v), and add 40% of it to the above-treated sodium hyaluronate alkaline solution;

[0052] Step 5): using 0.2% diatomaceous earth, 50° C., stirring, adsorption treatment for 40 min, and filtering;

[0053] Step 6): After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, the mixture was adsorbed with 0.3% activated carbon at 50°C with stirring for 50 minutes and filtered;

[0054] Step 7): The filtrate is concentrated using a nanofiltration membrane system, wherein the nanofiltration membrane has a molecular weight cutoff of 250 Da, an operating pressure of 20 bar, and a temperature controlled at 50° C. to obtain a nanofiltration concentrate, which is spray-dried to obtain a high-quality sodium hyaluronate sample with a full molecular weight distribution.

[0055] Example 3:

[0056] This embodiment provides a method for preparing sodium hyaluronate with a full molecular weight distribution, which specifically comprises the following steps:

[0057] Step 1): Take 1 kg of cosmetic grade sodium hyaluronate with a molecular weight of 1.2 million, spray 80 mL of 5% hydrogen peroxide evenly on the surface of the sodium hyaluronate material, and use 900 μW / cm 2 The dose was UV irradiated for 60 min;

[0058] Step 2): Dissolve the UV-irradiated sodium hyaluronate material in water at a concentration of 5% (w / v) and stir at room temperature until it is completely dissolved;

[0059] Step 3): adjusting the pH to 12 with a 5% (w / v) NaOH solution, and then subjecting the sample to ultrasonic treatment at 60 kHz for 60 min;

[0060] Step 4): Prepare a 0.6% (w / v) high molecular weight sodium hyaluronate solution of 1.2 million and add it to the treated sodium hyaluronate alkaline solution at a ratio of 40% (v / v);

[0061] Step 5): using 0.4% diatomaceous earth, stirring at 55° C., adsorption treatment for 40 min, and filtering;

[0062] Step 6): After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, the mixture was adsorbed with 0.3% activated carbon at 55°C with stirring for 50 minutes and filtered;

[0063] Step 7): The filtrate is concentrated using a nanofiltration membrane system, wherein the nanofiltration membrane has a molecular weight cutoff of 200 Da, an operating pressure of 30 bar, and a temperature controlled at 50° C. to obtain a nanofiltration concentrate, which is spray-dried to obtain a high-quality sodium hyaluronate sample with a full molecular weight distribution.

[0064] Example 4:

[0065] This embodiment provides a method for preparing sodium hyaluronate with a full molecular weight distribution, which specifically comprises the following steps:

[0066] Step 1): Take 1 kg of cosmetic grade sodium hyaluronate with a molecular weight of 1.4 million, and evenly spray 100 mL of 5% hydrogen peroxide on the surface of the sodium hyaluronate material. 2 The dose was UV irradiated for 60 min;

[0067] Step 2): Dissolve the UV-irradiated sodium hyaluronate in water at a concentration of 5% (w / v) and stir at room temperature until it is completely dissolved;

[0068] Step 3): The pH was adjusted to 12 using a 6% NaOH solution, and then the mixture was subjected to ultrasonic treatment at 90 kHz for 60 min;

[0069] Step 4): Prepare a 0.3% (w / v) high molecular weight sodium hyaluronate solution of 1.4 million and add 30% (v / v) of the solution to the treated sodium hyaluronate alkaline solution;

[0070] Step 5): using 0.6% diatomaceous earth, 55° C., stirring, adsorption treatment for 60 min, and filtering;

[0071] Step 6): After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, the mixture was adsorbed with 0.4% activated carbon at 55°C with stirring for 60 minutes and filtered;

[0072] Step 7): The filtrate is concentrated using a nanofiltration membrane system, wherein the nanofiltration membrane has a molecular weight cutoff of 200 Da, an operating pressure of 30 bar, and a temperature controlled at 50° C. to obtain a nanofiltration concentrate, which is spray-dried to obtain a high-quality sodium hyaluronate sample with a full molecular weight distribution.

[0073] Example 5:

[0074] This embodiment provides a method for preparing sodium hyaluronate with a full molecular weight distribution, which specifically comprises the following steps:

[0075] Step 1): Take 1 kg of cosmetic grade sodium hyaluronate with a molecular weight of 1.5 million, spray 100 mL of 5% hydrogen peroxide evenly on the surface of the sodium hyaluronate material, and use 1500 μW / cm 2 The dose was UV irradiated for 60 min;

[0076] Step 2): Dissolve the UV-irradiated sodium hyaluronate in water at a concentration of 3% (w / v) and stir at room temperature until it is completely dissolved;

[0077] Step 3): The pH was adjusted to 12 using a 5% NaOH solution, and then the mixture was subjected to ultrasonic treatment at 90 kHz for 60 min;

[0078] Step 4): Prepare a 1.5 million high molecular weight sodium hyaluronate solution with a concentration of 0.3% (w / v) and add it to the treated sodium hyaluronate alkaline solution at a ratio of 20% (v / v);

[0079] Step 5): using 0.8% diatomaceous earth, 55° C., stirring, adsorption treatment for 30 min, and filtering;

[0080] Step 6): After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, adsorb the mixture with 0.6% activated carbon at 55°C with stirring for 30 minutes and filter;

[0081] Step 7): The filtrate is concentrated using a nanofiltration membrane system, wherein the nanofiltration membrane has a molecular weight cutoff of 300 Da, an operating pressure of 30 bar, and a temperature controlled at 50° C. to obtain a nanofiltration concentrate, which is spray-dried to obtain a high-quality sodium hyaluronate sample with a full molecular weight distribution.

[0082] Example 6:

[0083] This embodiment provides a method for preparing sodium hyaluronate with a full molecular weight distribution, which specifically comprises the following steps:

[0084] Step 1): Take 1 kg of cosmetic grade sodium hyaluronate with a molecular weight of 2 million, and evenly spray 100 mL of 5% hydrogen peroxide on the surface of the sodium hyaluronate material. 2 The dose was UV irradiated for 60 min;

[0085] Step 2): Dissolve the UV-irradiated sodium hyaluronate in water at a concentration of 1% (w / v) and stir at room temperature until it is completely dissolved;

[0086] Step 3): adjusting the pH to 12 with a 5% NaOH solution, and then subjecting the sample to ultrasonic treatment at 100 kHz for 60 minutes;

[0087] Step 4): Prepare a 2 million high molecular weight sodium hyaluronate solution with a concentration of 0.1% (w / v), and add it to the treated sodium hyaluronate alkaline solution at a ratio of 30% (v / v);

[0088] Step 5): using 1% diatomaceous earth, stirring at 55° C., adsorption treatment for 50 min, and filtering;

[0089] Step 6): After adjusting the pH to 5.0 with 2% dilute sulfuric acid solution, adsorb the mixture with 1% activated carbon at 55°C with stirring for 50 minutes and filter;

[0090] Step 7): The filtrate is concentrated using a nanofiltration membrane system, wherein the nanofiltration membrane has a molecular weight cutoff of 300 Da, an operating pressure of 30 bar, and a temperature controlled at 50° C. to obtain a nanofiltration concentrate, which is spray-dried to obtain a high-quality sodium hyaluronate sample with a full molecular weight distribution.

[0091] Comparative Example 1:

[0092] This comparative example selected a commercially available sodium hyaluronate No. 1 (cosmetic grade, weight-average molecular weight 1.25 million).

[0093] Comparative Example 2:

[0094] This comparative example selected a commercially available sodium hyaluronate No. 2 (cosmetic grade, weight-average molecular weight 100,000).

[0095] Comparative Example 3:

[0096] The preparation method of this comparative example is substantially the same as that of Example 1, except that in step 1), only 50 mL of 1% hydrogen peroxide is evenly sprayed without UV irradiation, and the filtrate is no longer filtered and concentrated using a nanofiltration membrane system after activated carbon adsorption.

[0097] Comparative Example 4:

[0098] The preparation method of this comparative example is substantially the same as that of Example 1, except that only 300 μW / cm 2 The samples were irradiated with UV light for 60 min without using hydrogen peroxide treatment, and the filtrate was no longer filtered and concentrated using a nanofiltration membrane system after activated carbon adsorption.

[0099] Comparative Example 5:

[0100] In this comparative example, high molecular weight sodium hyaluronate with a weight average molecular weight of 2 million, sodium hyaluronate with a weight average molecular weight of 1 million, low molecular weight sodium hyaluronate with a weight average molecular weight of 300,000, and oligomeric sodium hyaluronate with a weight average molecular weight of less than 10,000 were mixed and stirred at a mass ratio of 1:1:1:1 for 60 minutes to obtain a sodium hyaluronate sample.

[0101] Example 7:

[0102] The different sodium hyaluronate solutions obtained in Examples 1-6 and Comparative Examples 3-5 were sampled at various locations to determine their uniformity and stability. Viscosities of various 1% sodium hyaluronate solutions were measured at 25°C using an NDJ-1 rotary viscometer with rotor No. 3 and 60 rpm. Each sample was stored at 4°C after preparation, and samples were taken for viscosity testing six months later. The results are shown in Table 1. The sodium hyaluronate obtained in Examples 1-6 exhibited a uniform appearance and good stability. However, the sodium hyaluronate obtained in Comparative Examples 3 and 4 exhibited a relatively uniform appearance but slightly poorer stability. Furthermore, the sodium hyaluronate exhibited higher viscosity, low filtration efficiency, and difficulty filtering using a nanofiltration membrane system. The sodium hyaluronate obtained in Comparative Example 5 exhibited poor uniformity and stability.

[0103] Table 1 Test results of uniformity and stability of sodium hyaluronate

[0104]

[0105] Example 8:

[0106] The molecular weight distribution of the sodium hyaluronate samples obtained in the above examples and comparative examples was measured, wherein the proportions of sodium hyaluronate molecules with weight average molecular weights Mw ranging from 2,000 to 10,000, 10,000 to 300,000, 300,000 to 1,000,000, and >1,000,000 are shown in Table 2.

[0107] Table 2 Detection results of molecular weight distribution of sodium hyaluronate

[0108]

[0109]

[0110] As can be seen from the data in Table 2, compared with commercially available sodium hyaluronate of different molecular weight specifications, the molecular weight distribution of the sodium hyaluronate raw materials obtained in Examples 1 to 6 is relatively wide, with a molecular weight dispersion coefficient Mw / Mn of greater than 5. Comparative Examples 3 and 4 have relatively narrow molecular weight distributions, with molecular weight dispersion coefficients below 2.5. This indicates that the effects of UV irradiation or hydrogen peroxide treatment alone are not ideal.

[0111] Example 9:

[0112] The endotoxin and foreign protein of the sodium hyaluronate prepared in Examples 1 to 6 were detected by the Limulus amebocyte lysate assay and the Folin phenol assay, respectively. The results are shown in Table 3:

[0113] Table 3 Hyaluronic acid quality inspection results

[0114] Example Endotoxin (EU) Miscellaneous Protein Example 1 0.045 <0.01% Example 2 0.031 <0.01% Example 3 0.043 <0.01% Example 4 0.032 <0.01% Example 5 0.026 <0.01% Example 6 0.028 <0.01% Comparative Example 1 0.13 2 0.09 Comparative Example 2 0.12 6 0.08 Comparative Example 3 0.12 5 0.08 Comparative Example 4 0.11 3 0.07 Comparative Example 5 0.14 2 0.11

[0115] From the results in Table 3, it can be seen that the hyaluronic acid obtained by the preparation method of Examples 1 to 6 has an endotoxin content of <0.01EU, a protein content of <0.01%, and an OD value at 280nm <0.01 and an OD value at 260nm <0.01, which shows higher biocompatibility and higher quality level.

[0116] Example 10:

[0117] 45 effective volunteers were selected, aged 20 to 55 years old. No products, including cosmetics and topical medicines, were allowed to be used on the test area for 2 to 3 days before the test. Before the test, the subjects cleaned the inner forearms of both hands and marked the 4×4 cm test area. Samples of formulas 1 to 8 (respectively, 1% of full molecular weight sodium hyaluronate in Examples 1 to 6, 1% of commercially available 1.2 million sodium hyaluronate in formula 7, and 1% of sodium hyaluronate in comparative example 5, with the remaining ingredients in the formula being 2% glycerol and 2% butylene glycol) were evenly applied and randomly distributed on the left and right arms at a dosage of 3 mg / cm 2 The skin moisture content of the test area was measured using a skin moisture meter before application and 30 minutes, 1 hour, 3 hours, and 6 hours after application. Each area was measured five times in parallel, and the average value was used as the test result. Each tester performed the same test on the same subject. All testers were kept in a room with constant temperature and humidity before and after the test. Skin moisture increase (%) = (water content after application - water content before application) / water content before application × 100%.

[0118] Table 4 Effect of hyaluronic acid on skin moisture

[0119] 30min1h3h6hFormula 132.2±1.9831.6±1.4525.0±1.6719.6±1.86Formula 233.3±2.1232.3±2.1227.8±2.0120.1±2.02Formula 332.3±1.2431.3±21.328.3±2.0821.3±1.39Formula 433.2±1.5332.6±1.8529.6±1.7521. 8±0.98Formula 5 32.3±2.12 31.4±1.86 28.7±1.78 21.4±1.57Formula 6 32.2±2.32 31.4±2.31 29.3±1.82 22.6±1.68Formula 7 27.1±1.35 25.2±1.62 21.4±1.25 16.7±1.72Formula 8 30.1±1.30 28.1±1.41 22.5±1.28 17.2±1.56

[0120] As shown in Table 3, the full molecular weight sodium hyaluronate prepared in the present invention has excellent moisturizing properties and is superior to commercially available sodium hyaluronate products. It can be used in cosmetics to exert a good moisturizing effect.

[0121] Example 11

[0122] The inflammatory response of human immortalized epidermal cells (HaCaT) induced by interleukin IL-1β was used as a model to verify that the sodium hyaluronate with full molecular weight distribution prepared by the present invention has an anti-inflammatory effect.

[0123] Human immortalized epidermal cells (HaCaT) were cultured at a rate of 5×10 3 cells / mL, 100 μL per well was inoculated in a 96-well plate, with 6 replicates per group. After culturing for 24 hours, the culture medium containing 10 ng / mL interleukin IL-1β and 20 mg / mL full molecular weight sodium hyaluronate of the present invention (Examples 1 to 6) and control sodium hyaluronate (Control Examples 1 to 5) was replaced. Cells without IL-1β and sodium hyaluronate were used as a normal control group, and cells with only IL-1β added without sodium hyaluronate were used as a model control group. After further culturing for 24 hours, the expression levels of interleukin IL-1α and tumor necrosis factor TNF-α were detected using an ELISA kit.

[0124] Table 5 Anti-inflammatory effect of sodium hyaluronate

[0125] Group IL-1α (ng / ml) TNF-α (ng / ml) Normal control 43.361.81 Model control 314.234.35 Example 1 102.822.14 Example 2 95.192.00 Example 3 92.172.07 Example 4 97.321.98 Example 5 82.832.05 Example 6 88.671.92 Comparative example 1 134.732.93 Comparative example 2 154.203.17 Comparative example 3 123.892.38 Comparative example 4 116.282.29 Comparative example 5 117.022.41

[0126] As can be seen from the data in Table 5, the hyaluronic acids of different embodiments and comparative examples can all reduce the expression levels of IL-1α and TNF-α in cells, and all show a certain anti-inflammatory effect. The sodium hyaluronate with a full molecular weight distribution prepared by the process of the present invention has a better anti-inflammatory effect than the ordinary sodium hyaluronate in the comparative example.

[0127] Example 12

[0128] Sodium dodecyl sulfate (SDS)-induced skin damage model was used as a model to verify the anti-cell damage and cell repair effects of the full molecular weight distribution sodium hyaluronate prepared in the present invention.

[0129] The EpiSkin skin model was transferred to a 12-well plate for culture. After culturing for 24 hours, the culture medium containing 0.5% (v / v) SDS was replaced. After incubation for 2 hours, an equal volume of culture medium containing 1% (w / v) full molecular weight sodium hyaluronate of the present invention (Examples 1 to 6) and control sodium hyaluronate (Control Examples 1 to 5) was directly added to each well to obtain a final concentration of 0.5% (w / v) sodium hyaluronate in the culture medium. Cells without SDS and sodium hyaluronate were used as a normal control group, and cells with only SDS but no sodium hyaluronate were used as a model control group. After further culture for 24 hours, the skin tissue model was removed, and the absorbance value was measured at 570 nm using a microplate reader using the MTT method. The tissue cell activity was calculated according to the following formula:

[0130] Tissue cell activity (%) = A p(t) / A n ×100%

[0131] Among them, A p(t) is the absorbance value of the positive control group or experimental group; A n is the absorbance value of the negative control group, and the results are as follows:

[0132] Table 6 Tissue cell activity after SDS and hyaluronic acid acted on skin model

[0133] Group Tissue Cell Activity (%) Normal Control 95.11 Model Control 54.58 Example 1 86.34 Example 2 88.16 Example 3 88.12 Example 4 89.57 Example 5 89.28 Example 6 92.78 Comparative Example 1 74.37 Comparative Example 2 6 819 Comparative Example 3 76.34 Comparative Example 4 72.28 Comparative Example 5 77.56

[0134] As shown in Table 6, the sodium hyaluronate with a full molecular weight distribution prepared by the present invention has a strong inhibitory effect on SDS-induced damage in the EpiSkin skin model and can effectively promote the repair of damaged cells. Compared with the conventional sodium hyaluronate in the comparative example, the sodium hyaluronate with a full molecular weight distribution prepared by the process of the present invention has a better effect on promoting cell repair.

[0135] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A method for preparing sodium hyaluronate with full molecular weight distribution, It is characterized in that The method comprises: Step 1): spraying the sodium hyaluronate solid raw material with hydrogen peroxide and irradiating it with ultraviolet light to obtain a sodium hyaluronate degradation material; Step 2): dissolving the sodium hyaluronate degradation material in water, and adjusting the pH to alkaline with a NaOH solution to obtain a sodium hyaluronate alkaline solution; Step 3): ultrasonically treating the sodium hyaluronate alkaline solution; Step 4): preparing the sodium hyaluronate solid raw material into a sodium hyaluronate solution with a concentration of 0.1% to 1% (w / v), and mixing it evenly with the sodium hyaluronate alkaline solution after ultrasound obtained in step 3) at an addition ratio of 20% to 60% (v / v); Step 5): Adsorbing with diatomaceous earth and activated carbon respectively, filtering and concentrating with a nanofiltration membrane, and drying to obtain the sodium hyaluronate with full molecular weight distribution.

2. The method for preparing sodium hyaluronate with full molecular weight distribution according to claim 1, It is characterized in that In the step 1), 50 to 100 mL of hydrogen peroxide with a mass concentration of 1% to 5% is sprayed per kilogram of the sodium hyaluronate solid raw material, and the ultraviolet irradiation dose is 300 to 1500 μW / cm 2 The irradiation time is 50 to 70 minutes.

3. The method for preparing sodium hyaluronate with full molecular weight distribution according to claim 1, It is characterized in that The molecular weight of the sodium hyaluronate solid raw material is 1 to 2 million.

4. The method for preparing sodium hyaluronate with full molecular weight distribution according to claim 3, It is characterized in that The sodium hyaluronate solid raw material used is selected from one or more of cosmetic grade sodium hyaluronate, food grade sodium hyaluronate and pharmaceutical grade sodium hyaluronate.

5. The method for preparing sodium hyaluronate with full molecular weight distribution according to claim 1, It is characterized in that In the step 2), the volume concentration of the sodium hyaluronate degradation material after being dissolved in water is 1% to 10% (w / v); and / or, Adjust the pH to 10-12 with NaOH solution.

6. The method for preparing sodium hyaluronate with full molecular weight distribution according to claim 1, It is characterized in that In the step 3), the frequency of the ultrasonic treatment is 10 to 100 kHz, and the time is 15 to 180 minutes.

7. The method for preparing sodium hyaluronate with full molecular weight distribution according to claim 1, It is characterized in that In the step 5), The step of using diatomaceous earth adsorption treatment includes: adding 0.1% to 1% (w / v) diatomaceous earth based on the mass of the sodium hyaluronate alkaline solution, stirring and adsorbing at 45-80° C. for 30 to 60 minutes, and filtering; and / or, The steps of using activated carbon adsorption treatment include: adjusting the pH to 6-7 with a dilute acid solution, adding 0.1%-1% (w / v) activated carbon based on the mass of the sodium hyaluronate solution, stirring and adsorbing at 45-80° C. for 30-60 minutes, and filtering; preferably, the dilute acid solution is selected from one or more of a dilute hydrochloric acid solution, a dilute sulfuric acid solution, a dilute acetic acid solution, and a dilute hypochlorous acid solution.

8. The method for preparing sodium hyaluronate with full molecular weight distribution according to claim 1, It is characterized in that In the step 5), the molecular weight cut-off of the nanofiltration membrane is 200-300 Da, the operating pressure is controlled at 15-30 bar, and the temperature is controlled at 30-50° C.

9. Sodium hyaluronate with full molecular weight distribution obtained by the preparation method according to any one of claims 1 to 8, It is characterized in that The weight average molecular weight of the sodium hyaluronate with full molecular weight distribution ranges from 2,000 to 1.5 million, and the molecular weight dispersion coefficient Mw / Mn is above 5; Preferably, the endotoxin content in the sodium hyaluronate is <0.01EU, and the protein content is <0.01%; Preferably, the OD value of the sodium hyaluronate at 280 nm is <0.01, and the OD value at 260 nm is <0.

01.

10. Use of full molecular weight sodium hyaluronate obtained by the preparation method according to any one of claims 1 to 8 in the preparation of moisturizers, lubricants, anti-inflammatory agents and / or cell repair agents for medical, food and / or cosmetic use.