Preparation method and application of medical grade agarose with different molecular weights

Abstract

The invention provides a preparation method and application of medical grade agarose with different molecular weights, and belongs to the technical field of biomedical materials. According to the invention, the crude agarose product is treated by DMSO, EDTA-Na2 and DEAE-cellulose, and then the acid concentration, the acid treatment temperature, the acid treatment time and the like in the acid treatment stage are finely regulated, so that the medical-grade agarose with medium-high molecular weight (3000-100000), medium molecular weight (3000-3000) and low molecular weight (1t, 3000) is successfully prepared. The medium-high molecular weight and medium molecular weight medical grade agarose provided by the invention has excellent gel performance, adjustable mechanical strength and injection performance, and is suitable for the fields of skin implants, gel dressings, tissue engineering and the like; the low-molecular-weight medical grade agarose shows good water solubility, fluidity and oxidation resistance, and has significant application potential in the fields of medical skin care products and functional dressings.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a preparation method and application of medical-grade agarose with different molecular weights. Background Art

[0002] The skin is an important and complex protective barrier of the human body, with multiple physiological functions, including defending against the invasion of external pathogens and harmful substances, maintaining the dynamic balance of body water and electrolytes, regulating immune responses, and also playing a buffering and protective role under external environmental pressure. When the skin is traumatized or diseased, its barrier function is significantly weakened, triggering a series of pathological changes, such as an increased risk of infection, exacerbated inflammatory responses, and impaired tissue repair ability, seriously threatening the physiological health of patients. In addition, skin injuries can also have a negative impact on the psychological state of patients, potentially inducing mood disorders such as anxiety and depression, and significantly reducing the quality of life. The pathological consequences of skin trauma may include scar formation, premature skin aging, and even the occurrence of malignant lesions such as skin cancer.

[0003] In recent years, with the rapid development of regenerative medicine and bioengineering technologies, the application of biomedical materials in the field of skin repair and regeneration has received extensive attention. As a natural polysaccharide material extracted from red algae, agarose has become a research hotspot in biomedical materials due to its excellent biocompatibility, non-immunogenicity, and controllable thermoreversible gel properties. Its chemical structure consists of β-D-galactose and 3,6-anhydro-α-L-galactose, and it can dissolve at high temperatures and form a thermoreversible gel upon cooling. Agarose materials have adjustable mechanical properties and good degradation characteristics, and have been widely used in the fields of cell culture, drug delivery carriers, and tissue engineering scaffolds.

[0004] However, currently, commercially available agarose mainly consists of high-molecular-weight products, and its high gel strength and low fluidity limit its application in fields such as skin dressings, implants, and skin care products. In contrast, low-molecular-weight agarose has been preliminarily applied in fields such as food, daily chemicals, and aquaculture due to its high water solubility and biological activity. However, low-molecular-weight agarose degrades rapidly and has poor mechanical support, making it difficult to meet the application requirements of implants and functional gel dressings in skin medicine.

[0005] Currently, the main method for preparing low-molecular-weight agarose is enzymatic hydrolysis, but this method faces a series of technical difficulties, such as insufficient agarose enzyme activity, harsh enzyme reaction conditions, high cost of enzyme preparations, and easy inactivation of enzymes, which limit its industrial production capacity. In contrast, the acid hydrolysis method has attracted attention due to its advantages such as simple process, low cost, and easy availability of raw materials. However, the existing acid hydrolysis method has problems of poor molecular weight control accuracy and insufficient product purity, making it difficult to meet the stringent requirements for material quality in skin medicine applications. To address the above problems, there is an urgent need to develop an efficient, controllable, and industrially scalable agarose preparation method to achieve precise regulation of molecular weight and meet the needs of various skin medicine products. Summary of the Invention

[0006] Aiming at the above deficiencies of the prior art, the present invention provides a method for preparing medical-grade agarose with different molecular weights and its applications. The present invention processes agarose crude products with DMSO, EDTA-Na2, and DEAE-cellulose, and then successfully prepares medium-high molecular weight (molecular weight 30,000 - 100,000), medium molecular weight (3,000 - 30,000), and low molecular weight (molecular weight < 3,000) medical-grade agarose by finely regulating the acid concentration, acid treatment temperature, and time in the acid treatment stage. The research results of the present invention show that medium-high molecular weight and medium molecular weight medical-grade agarose have excellent gel properties, adjustable mechanical strength, and injection properties, and are suitable for fields such as skin implants, gel dressings, and tissue engineering; low molecular weight medical-grade agarose exhibits good water solubility, fluidity, and antioxidant capacity, and has significant application potential in the fields of medical skin care products and functional dressings.

[0007] To achieve the above object, the specific technical solutions of the present invention are as follows:

[0008] In the first aspect, the present invention provides a method for preparing medical-grade agarose with different molecular weights, including the following steps: mixing agarose crude product with dimethyl sulfoxide and heating, centrifuging, and taking the supernatant to obtain intermediate I; mixing intermediate I with EDTA-Na2 solution and heating, filtering, and taking the filter cake to obtain intermediate II; formulating intermediate II into a solution, adding DEAE-cellulose, heating, centrifuging, and taking the supernatant to obtain intermediate III; formulating intermediate III into agarose hydrogel and treating it with hydrochloric acid to obtain medical-grade agarose with different molecular weights.

[0009] Furthermore, the method for preparing medical-grade agarose with different molecular weights includes the following steps:

[0010] (1) Mix agarose crude product with dimethyl sulfoxide (DMSO), heat-treat at 65 - 85 °C, centrifuge, and dry the supernatant after gelling to obtain intermediate I;

[0011] (2) Mix the intermediate I obtained in step (1) with the EDTA-Na2 solution, heat-treat at 45-95 °C, filter, and dry the filter cake to obtain intermediate II;

[0012] (3) Prepare the intermediate II obtained in step (2) into a solution, add DEAE-cellulose, heat-treat at 60-99 °C, centrifuge, and dry the supernatant after gelling to obtain intermediate III;

[0013] (4) Prepare the intermediate III obtained in step (3) into an agarose hydrogel, add a hydrochloric acid solution with a concentration of 0.2 M - 1.5 M for treatment, the treatment temperature is 25-90 °C, the time is 2-10 h, rinse until the pH of the washing liquid is neutral after the treatment, and dry to obtain medical-grade agarose with different molecular weights.

[0014] Further, in order to improve the quality of the prepared medical-grade agarose, steps (1)-(3) are each carried out twice, that is, step (1) is continuously operated twice and then step (2) is carried out; step (2) is continuously operated twice and then step (3) is carried out; step (3) is continuously operated twice and then step (4) is carried out.

[0015] Further, the molecular weight of the crude agarose is greater than 100,000.

[0016] Further, the mass-volume ratio of the crude agarose to dimethyl sulfoxide in step (1) is 1:(40-60) g / mL.

[0017] Further, the heating treatment time in step (1) is 6-12 h.

[0018] Further, the mass fraction of the EDTA-Na2 solution in step (2) is 2-8 g / L.

[0019] Even further, the mass fraction of the first EDTA-Na2 treatment in step (2) is 6 g / L, and the mass fraction of the second EDTA-Na2 treatment is 4 g / L.

[0020] Further, the heating treatment time in step (2) is 2-6 h.

[0021] Even further, the heating treatment temperature in step (2) is 55 °C and the time is 4 h.

[0022] Further, the mass of the DEAE-cellulose in step (3) is 1-7 times the mass of the crude agarose.

[0023] Further, in step (3), the first dosage of DEAE-cellulose is 7 times the mass of the crude agarose, and the second dosage of DEAE-cellulose is 5 times the mass of the crude agarose.

[0024] Further, in step (3), the time of the heat treatment is 1 - 5 h.

[0025] Further still, in step (3), the temperature of the heat treatment is 70 - 90 °C and the time is 1 - 3 h.

[0026] Further, in step (4), when the concentration of the hydrochloric acid is 0.2 M - 1.5 M, the treatment temperature is 25 - 40 °C, and the time is 8 - 10 h, medium and high molecular weight medical-grade agarose with a molecular weight of 30,000 to 100,000 is obtained;

[0027] when the concentration of the hydrochloric acid is 0.4 M - 1.5 M, the treatment temperature is 55 - 65 °C, and the time is 7 - 9 h, medium molecular weight medical-grade agarose with a molecular weight of 3,000 to 30,000 is obtained;

[0028] when the concentration of the hydrochloric acid is 1.2 M - 1.5 M, the treatment temperature is 70 - 90 °C, and the time is 2 - 6 h, low molecular weight medical-grade agarose with a molecular weight of less than 3,000 is obtained.

[0029] In a second aspect, the present invention provides medical-grade agarose of different molecular weights prepared by the said method.

[0030] In a third aspect, the present invention provides the application of the said medical-grade agarose of different molecular weights in the biomedical field, including: medium and high molecular weight and medium molecular weight medical-grade agarose are used for implants and gel dressings; low molecular weight medical-grade agarose is used for anti-aging skin care products and skin repair products.

[0031] Specifically, in anti-aging skin care products, the high molecular weight medical-grade agarose forms a protective barrier; the medium molecular weight medical-grade agarose improves skin elasticity; the low molecular weight medical-grade agarose promotes cell regeneration and improves skin texture.

[0032] Specifically, in skin repair products, the medical-grade agarose of different molecular weights all has a moisturizing effect; the medium molecular weight and low molecular weight medical-grade agarose have a significant antioxidant effect; the low molecular weight medical-grade agarose can accelerate the repair of damaged tissues.

[0033] In a fourth aspect, the present invention provides a comprehensive preparation suitable for dermatology, including the said medical-grade agarose of different molecular weights, and the proportion of the medical-grade agarose of each molecular weight in the preparation is adjusted according to actual needs to achieve the synergistic effect of moisturizing, antioxidant and repair functions.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) The present invention provides a method for preparing medical-grade agarose with different molecular weights. The method of the present invention uses crude agarose with a molecular weight > 100,000 as the raw material, which is treated with DMSO, EDTA-Na2, and DEAE-cellulose and then treated with hydrochloric acid. By adjusting the concentration of hydrochloric acid, treatment temperature, and time, medical-grade agarose with medium-high molecular weights in the range of 30,000 to 100,000, medical-grade agarose with medium molecular weights in the range of 3,000 to 30,000, and medical-grade agarose with low molecular weights less than 3,000 can be obtained.

[0036] (2) The medical-grade agarose with different molecular weights prepared by the method of the present invention has the advantages of high purity and low impurity content. Among them, the sulfate content ≤ 0.12%.

[0037] (3) The lower the molecular weight of the agarose prepared by the method of the present invention, the lower its gel strength and viscosity, the faster the degradation rate, and at the same time, it has better injectability and antioxidant ability.

[0038] (4) The medical-grade agarose with different molecular weights prepared by the method of the present invention all exhibit excellent moisturizing effects and repair functions, and have significant application potential in the fields of medical skin care products and functional dressings. Description of the Drawings

[0039] Figure 1 is a physical diagram of medical-grade agarose with different molecular weights;

[0040] Figure 2 is the infrared of medical-grade agarose with different molecular weights;

[0041] Figure 3 is the gel strength of medical-grade agarose with different molecular weights;

[0042] Figure 4 is the viscosity of medical-grade agarose with different molecular weights;

[0043] Figure 5 is the injectability of medical-grade agarose with different molecular weights;

[0044] Figure 6 is the degradability of medical-grade agarose with different molecular weights;

[0045] Figure 7 is the antioxidant ability of medical-grade agarose with different molecular weights;

[0046] Figure 8 is the zebrafish moisturizing effect of medical-grade agarose with different molecular weights;

[0047] Figure 9 The repair effects of medical-grade agarose with different molecular weights on zebrafish. Detailed implementation manners

[0048] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0049] The present invention provides a preparation method of medical-grade agarose with different molecular weights, including the following steps:

[0050] (1) Mix the crude agarose with dimethyl sulfoxide (DMSO) at a mass-to-volume ratio of g / mL of 1:(40 - 60), heat-treat at 65 - 85 °C for 6 - 12 h, centrifuge, and dry the supernatant after gelling to obtain intermediate I;

[0051] (2) Mix the intermediate I obtained in step (1) with an EDTA-Na2 solution, heat-treat at 45 - 95 °C for 2 - 6 h, filter, and dry the filter cake to obtain intermediate II;

[0052] (3) Prepare the intermediate II obtained in step (2) into a solution, add DEAE-cellulose with a mass 1 - 7 times that of the crude agarose, heat-treat at 60 - 99 °C for 1 - 5 h, centrifuge, and dry the supernatant after gelling to obtain intermediate III;

[0053] (4) Prepare the intermediate III obtained in step (3) into an agarose hydrogel, add a 0.2 M - 1.5 M hydrochloric acid solution for treatment, with the treatment temperature being 25 - 90 °C and the time being 2 - 10 h. After the treatment, rinse until the pH of the washing solution is neutral, and dry to obtain medical-grade agarose with different molecular weights.

[0054] In the following specific examples, in order to improve the quality of the prepared medical-grade agarose, steps (1) - (3) are each carried out twice, that is, step (1) is continuously operated twice and then enters step (2); step (2) is continuously operated twice and then enters step (3); step (3) is continuously operated twice and then enters step (4).

[0055] The molecular weight of the crude agarose is greater than 100,000. It can be obtained by conventional purchase or by extraction / preparation through existing technologies. In the following specific examples, the crude agarose is obtained by extracting from Gelidium amansii.

[0056] In some examples, the mass fraction of the EDTA-Na2 solution in step (2) is 2 - 8 g / L.

[0057] In some examples, in step (4), when the concentration of the hydrochloric acid is 0.2 M to 1.5 M, the treatment temperature is 25 to 40 °C, and the time is 8 to 10 h, medium- and high-molecular-weight medical-grade agarose with a molecular weight of 30,000 to 100,000 is obtained; when the concentration of the hydrochloric acid is 0.4 M to 1.5 M, the treatment temperature is 55 to 65 °C, and the time is 7 to 9 h, medium-molecular-weight medical-grade agarose with a molecular weight of 3,000 to 30,000 is obtained; when the concentration of the hydrochloric acid is 1.2 M to 1.5 M, the treatment temperature is 70 to 90 °C, and the time is 2 to 6 h, low-molecular-weight medical-grade agarose with a molecular weight less than 3,000 is obtained.

[0058] In the following specific examples, the method for measuring the sulfate content uses the barium sulfate turbidimetry, and the steps are as follows:

[0059] (1) Preparation of potassium sulfate standard solution: K2SO4 is dried at 105 °C for 2 h. After cooling to room temperature, 0.1088 g is weighed and made up to 100 mL with 1 mol / L hydrochloric acid, and stored for later use (sulfate ion concentration 0.6 mg / mL).

[0060] (2) Preparation of 3% trichloroacetic acid solution: Weigh 3 g of trichloroacetic acid, dissolve it with ultrapure water and make up to 100 mL, and store for later use.

[0061] (3) Preparation of 0.5% gelatin solution: Weigh 0.5 g of gelatin, dissolve it ultrasonically with ultrapure water and make up to 100 mL, and store for later use.

[0062] (4) Preparation of gelatin-barium chloride solution: Weigh 0.5 g of BaCl2, dissolve it ultrasonically with 100 mL of 0.5% gelatin solution, and store for later use.

[0063] (5) Preparation of sample solution: Weigh 400 mg of the sample in a round-bottom flask, add 50 mL of 1 mol / L hydrochloric acid, and hydrolyze it in a water bath at 105 °C for 4 h. After the sample is cooled, make it up to 50 mL with 1 mol / L hydrochloric acid, decolorize it and filter to obtain a sample solution with a concentration of 8 mg / mL.

[0064] (6) Plotting of the standard curve: Accurately pipette 0.02, 0.06, 0.10, 0.14, 0.18, and 0.20 mL of the potassium sulfate standard solution, make up to 0.2 mL with 1 mol / L hydrochloric acid. Using the HC1 solution as the blank, add 3.8 mL of 3% trichloroacetic acid solution and 1.0 mL of barium chloride solution respectively, shake well, let stand for 15 min, measure the absorbance at a wavelength of 360 nm. Plot a graph with the number of micrograms of sulfate ion as the abscissa and the absorbance as the ordinate to obtain the standard curve. In the examples and comparative examples, the potassium sulfate standard solution was diluted 6 times, and the standard curve equation was Y = 0.0169X - 0.0055.

[0065] (7) Determination of the sulfate content in the test solution: Accurately pipette 0.20 mL of the test solution, add 3.8 mL of 3% trichloroacetic acid solution and 1.0 mL of barium chloride solution, shake well, let stand for 15 min, measure the absorbance at a wavelength of 360 nm, and calculate the sulfate content in the test solution using the standard curve.

[0066] In the following specific examples, the molecular weight of the agarose was determined by gel permeation chromatography and the Ubbelohde capillary viscometer method. The gel permeation chromatograph, model Optilab T-rEX and 1260, manufacturer WYATT, was used in conjunction with an 18-angle laser light scattering detector. In the Ubbelohde capillary viscometer method, the Mark-Houwink equation was used to determine the molecular weight of each agarose sample, [η] = 0.07M 0.72 , where [η] is the intrinsic viscosity (mL / g).

[0067] In the following specific examples, unless otherwise specified, the raw materials or reagents used were obtained by conventional purchase.

[0068] Example 1 A medical-grade agarose with different molecular weights

[0069] 1. Extraction of high-molecular-weight Gelidium amansii agarose

[0070] Pretreat Gracilaria lemaneiformis, wash it, and dry it with blowing air for later use; then conduct alkali treatment. Add 30 g of dried Gracilaria lemaneiformis to 600 mL of 8% NaOH solution, treat it at 75 °C for 3 h, pour off the liquid, and soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then conduct acid treatment. Add 600 mL of 0.01 mol / L acetic acid solution to the Gracilaria lemaneiformis after alkali treatment, pour off the liquid after treating for 30 min, and then soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then conduct bleaching treatment. Add 600 mL of sodium hypochlorite solution with an available chlorine mass fraction of 0.05% to the Gracilaria lemaneiformis after acid treatment, bleach it for 30 min, and then rinse it with ultrapure water until the pH of the washing liquid is neutral. Finally, add the bleached Gracilaria lemaneiformis to ultrapure water, heat and extract it at 110 °C, filter it while it is hot, cool and solidify it at room temperature, dehydrate it by freeze-thawing, and then dry it to obtain crude agarose with a molecular weight greater than 100,000.

[0071] 2. Two consecutive DMSO treatments

[0072] Mix the crude agarose with DMSO (crude agarose:DMSO = 1 g:50 mL) at 75 °C, stir it in an oil bath for 8 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and crush the obtained dry gel to obtain Intermediate I.

[0073] 3. Two consecutive EDTA-Na2 treatments

[0074] Add Intermediate I to the EDTA-Na2 solution (the concentration for the first EDTA-Na2 treatment is 6 g / L, and the concentration for the second EDTA-Na2 treatment is 4 g / L) at 55 °C, stir it for 4 h, then filter it and wash away the residual EDTA-Na2, and dry it to obtain Intermediate II.

[0075] 4. Two consecutive DEAE-cellulose treatments

[0076] Prepare Intermediate II into a 2% solution, dissolve it at 100 °C, cool it to 80 °C, add DEAE-cellulose (the dosage for the first DEAE-cellulose treatment is 7 times the mass of the crude agarose, and the dosage for the second treatment is 5 times the mass of the crude agarose), and stir it at 80 °C for 1.5 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and finally crush the obtained dry gel to obtain Intermediate III.

[0077] 5.1 Preparation of high molecular weight medical grade agarose

[0078] At 100 °C, Intermediate III was dissolved in ultrapure water and stirred continuously for 3 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 0.2 M HCl solution was added to the hydrogel pieces and treated at 37 °C for 10 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and crushed to obtain medium- and high-molecular-weight medical-grade agarose powder.

[0079] 5.2 Preparation of Medium-Molecular-Weight Medical-Grade Agarose

[0080] At 100 °C, Intermediate III was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 1.2 M HCl solution was added to the hydrogel pieces and treated at 60 °C for 8 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and crushed to obtain medium-molecular-weight medical-grade agarose powder.

[0081] 5.3 Preparation of Low-Molecular-Weight Medical-Grade Agarose

[0082] At 100 °C, the extracted high-molecular-weight Gelidium amansii agarose powder was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 1.5 M hydrochloric acid solution was added to the hydrogel pieces and treated at 80 °C for 2 h to obtain a low-molecular-weight medical-grade agarose solution. The obtained low-molecular-weight medical-grade agarose solution was dialyzed until the pH was neutral, freeze-dried and crushed to obtain low-molecular-weight medical-grade agarose powder.

[0083] Example 2 A Medical-Grade Agarose with Different Molecular Weights

[0084] 1. Extraction of High-Molecular-Weight Gelidium amansii Agarose

[0085] Pretreat Gelidium amansii, wash it and then dry it with blowing air for standby; subsequently, perform alkali treatment. Add 30 g of the dried Gelidium amansii into 600 mL of 8% NaOH solution, treat it at 75 °C for 3 h, pour out the liquid, and soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then perform acid treatment. Add 600 mL of 0.01 mol / L acetic acid solution to the Gelidium amansii after alkali treatment, pour out the liquid after treating for 30 min, and then soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then perform bleaching treatment. Add 600 mL of sodium hypochlorite solution with an available chlorine mass fraction of 0.05% to the Gelidium amansii after acid treatment, bleach it for 30 min, and then rinse it with ultrapure water until the pH of the washing liquid is neutral. Finally, add the bleached Gelidium amansii into ultrapure water, heat and extract it at 110 °C, filter it while it is hot, cool and solidify it at room temperature, dehydrate it by freeze-thawing, and then dry it to obtain crude agarose with a molecular weight greater than 100,000.

[0086] 2. Two consecutive DMSO treatments

[0087] Mix the crude agarose with DMSO (crude agarose:DMSO = 1 g:50 mL) at 75 °C, stir it in an oil bath for 8 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and crush the obtained dry gel to obtain intermediate I.

[0088] 3. Two consecutive EDTA-Na2 treatments

[0089] Add intermediate I into the EDTA-Na2 solution (the concentration for the first EDTA-Na2 treatment is 6 g / L, and the concentration for the second EDTA-Na2 treatment is 4 g / L) at 50 °C, stir it for 5 h, then filter it and wash away the residual EDTA-Na2, and dry it to obtain intermediate II.

[0090] 4. Two consecutive DEAE-cellulose treatments

[0091] Prepare intermediate II into a 2% solution, dissolve it at 100 °C, cool it to 80 °C, add DEAE-cellulose (the dosage for the first DEAE-cellulose treatment is 7 times the mass of the crude agarose, and the dosage for the second treatment is 5 times the mass of the crude agarose), and stir it at 80 °C for 1.5 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and finally crush the obtained dry gel to obtain intermediate III.

[0092] 5.1 Preparation of high molecular weight medical grade agarose

[0093] At 100 °C, Intermediate III was dissolved in ultrapure water and stirred continuously for 3 min. Then, the solution was left standing at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 0.4 M HCl solution was added to the hydrogel pieces and treated at 37 °C for 10 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and pulverized to obtain medium- and high-molecular-weight medical-grade agarose powder.

[0094] Preparation of medium-molecular-weight medical-grade agarose

[0095] At 100 °C, Intermediate III was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was left standing at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 1.2 M HCl solution was added to the hydrogel pieces and treated at 60 °C for 8 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and pulverized to obtain medium-molecular-weight medical-grade agarose powder.

[0096] Preparation of low-molecular-weight medical-grade agarose

[0097] At 100 °C, the extracted high-molecular-weight Gelidium amansii agarose powder was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was left standing at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 1.5 M hydrochloric acid solution was added to the hydrogel pieces and treated at 90 °C for 2 h to obtain a low-molecular-weight medical-grade agarose solution. The obtained low-molecular-weight medical-grade agarose solution was dialyzed until the pH was neutral, freeze-dried and pulverized to obtain low-molecular-weight medical-grade agarose powder.

[0098] Example 3 A medical-grade agarose with different molecular weights

[0099] 1. Extraction of high-molecular-weight Gelidium amansii agarose

[0100] Pretreat Gracilaria lemaneiformis, wash it and dry it with blowing air for standby; then perform alkali treatment. Add 30 g of dried Gracilaria lemaneiformis into 600 mL of 8% NaOH solution, treat it at 75 °C for 3 h, pour out the liquid and soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then perform acid treatment. Add 600 mL of 0.01 mol / L acetic acid solution to the Gracilaria lemaneiformis after alkali treatment, pour out the liquid after treating for 30 min, and then soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then perform bleaching treatment. Add 600 mL of sodium hypochlorite solution with an available chlorine mass fraction of 0.05% to the Gracilaria lemaneiformis after acid treatment, bleach it for 30 min and then rinse it with ultrapure water until the pH of the washing liquid is neutral. Finally, add the bleached Gracilaria lemaneiformis into ultrapure water, heat and extract it at 110 °C, filter it while it is hot, cool and solidify it at room temperature, dehydrate it by freeze-thawing and then dry it to obtain crude agarose with a molecular weight greater than 100,000.

[0101] 2. Two consecutive DMSO treatments

[0102] Mix the crude agarose with DMSO (crude agarose:DMSO = 1 g:60 mL) at 75 °C, stir it in an oil bath for 8 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and crush the obtained dry gel to obtain Intermediate I.

[0103] 3. Two consecutive EDTA-Na2 treatments

[0104] Add Intermediate I into the EDTA-Na2 solution (the concentration for the first EDTA-Na2 treatment is 6 g / L and the concentration for the second EDTA-Na2 treatment is 4 g / L) at 50 °C and stir for 5 h, then filter it and wash away the residual EDTA-Na2, and dry it to obtain Intermediate II.

[0105] 4. Two consecutive DEAE-cellulose treatments

[0106] Prepare Intermediate II into a 2% solution, dissolve it at 100 °C, cool it to 80 °C, add DEAE-cellulose (the dosage for the first DEAE-cellulose treatment is 7 times the mass of the crude agarose, and the dosage for the second treatment is 5 times the mass of the crude agarose), and stir it at 80 °C for 1.5 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and finally crush the obtained dry gel to obtain Intermediate III.

[0107] 5.1 Preparation of high molecular weight medical grade agarose

[0108] At 100 °C, intermediate III was dissolved in ultrapure water and stirred continuously for 3 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into blocks of about 1 cm x 1 cm, and 0.4 M HCl solution was added to the hydrogel blocks, and treated at 37 °C for 10 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and pulverized to obtain medium- and high-molecular-weight medical-grade agarose powder.

[0109] Preparation of medium-molecular-weight medical-grade agarose

[0110] At 100 °C, intermediate III was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into blocks of about 1 cm x 1 cm, and 1.5 M HCl solution was added to the hydrogel blocks, and treated at 60 °C for 7 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and pulverized to obtain medium-molecular-weight medical-grade agarose powder.

[0111] Preparation of low-molecular-weight medical-grade agarose

[0112] At 100 °C, the extracted high-molecular-weight Gelidium amansii agarose powder was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into blocks of about 1 cm x 1 cm, and 1.5 M hydrochloric acid solution was added to the hydrogel blocks, and treated at 90 °C for 2 h to obtain a low-molecular-weight medical-grade agarose solution. The obtained low-molecular-weight medical-grade agarose solution was dialyzed until the pH was neutral, freeze-dried and pulverized to obtain low-molecular-weight medical-grade agarose powder.

[0113] Example 4 A medical-grade agarose with different molecular weights

[0114] 1. Extraction of high-molecular-weight Gelidium amansii agarose

[0115] Pretreat Gracilaria, wash it and dry it with blowing air for later use; then conduct alkali treatment. Add 30 g of dried Gracilaria into 600 mL of 8% NaOH solution, treat it at 75 °C for 3 h, pour out the liquid, and soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then conduct acid treatment. Add 600 mL of 0.01 mol / L acetic acid solution to the Gracilaria after alkali treatment, pour out the liquid after treating for 30 min, and then soak and rinse it with ultrapure water until the pH of the washing liquid is neutral; then conduct bleaching treatment. Add 600 mL of sodium hypochlorite solution with an available chlorine mass fraction of 0.05% to the Gracilaria after acid treatment, bleach it for 30 min, and then rinse it with ultrapure water until the pH of the washing liquid is neutral. Finally, add the bleached Gracilaria into ultrapure water, heat and extract it at 110 °C, filter it while it is hot, cool and solidify it at room temperature, dehydrate it by freeze-thawing, and then dry it to obtain crude agarose with a molecular weight greater than 100,000.

[0116] 2. Two consecutive DMSO treatments

[0117] Mix the crude agarose with DMSO (crude agarose:DMSO = 1 g:50 mL) at 75 °C, stir it in an oil bath for 8 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and crush the obtained dry gel to obtain intermediate I.

[0118] 3. Two consecutive EDTA-Na2 treatments

[0119] Add intermediate I into the EDTA-Na2 solution (the concentration for the first EDTA-Na2 treatment is 6 g / L, and the concentration for the second EDTA-Na2 treatment is 4 g / L) at 50 °C and stir for 5 h, then filter it and wash away the residual EDTA-Na2, and dry it to obtain intermediate II.

[0120] 4. Two consecutive DEAE-cellulose treatments

[0121] Prepare intermediate II into a 2% solution, dissolve it at 100 °C, cool it to 80 °C, add DEAE-cellulose (the dosage for the first DEAE-cellulose treatment is 7 times the mass of the crude agarose, and the dosage for the second treatment is 5 times the mass of the crude agarose), and stir it at 80 °C for 2 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and finally crush the obtained dry gel to obtain intermediate III.

[0122] 5.1 Preparation of high molecular weight medical grade agarose

[0123] At 100 °C, intermediate III was dissolved in ultrapure water and stirred continuously for 3 min. Then, the solution was left standing at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 0.4 M HCl solution was added to the hydrogel pieces and treated at 37 °C for 10 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and pulverized to obtain medium- and high-molecular-weight medical-grade agarose powder.

[0124] Preparation of medium-molecular-weight medical-grade agarose

[0125] At 100 °C, intermediate III was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was left standing at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 1.3 M HCl solution was added to the hydrogel pieces and treated at 65 °C for 8 h to obtain a medium- and high-molecular-weight medical-grade agarose hydrogel. The obtained medium- and high-molecular-weight medical-grade agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, freeze-dried and pulverized to obtain medium-molecular-weight medical-grade agarose powder.

[0126] Preparation of low-molecular-weight medical-grade agarose

[0127] At 100 °C, the extracted high-molecular-weight Gelidium amansii agarose powder was dissolved in ultrapure water and stirred continuously for 30 min. Then, the solution was left standing at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 1.2 M hydrochloric acid solution was added to the hydrogel pieces and treated at 80 °C for 3 h to obtain a low-molecular-weight medical-grade agarose solution. The obtained low-molecular-weight medical-grade agarose solution was dialyzed until the pH was neutral, freeze-dried and pulverized to obtain low-molecular-weight medical-grade agarose powder.

[0128] Comparative Example 1

[0129] 1. Two consecutive DMF treatments

[0130] At 75 °C, the crude agarose obtained in Step 1 of Example 1 was mixed with DMF (N,N-dimethylformamide) (crude agarose:DMF = 1 g:50 mL), stirred in an oil bath for 8 h, centrifuged to remove the precipitate, and the supernatant was gelled at room temperature and then dried. The obtained dry gel was pulverized to obtain intermediate I.

[0131] 2. Two consecutive EDTA-Na2 treatments

[0132] The intermediate I was added to the EDTA-Na2 solution at 55 °C (the concentration of the first EDTA-Na2 treatment was 6 g / L, and the concentration of the second EDTA-Na2 treatment was 4 g / L), stirred for 4 h, then filtered by suction and the residual EDTA-Na2 was washed away, and dried to obtain intermediate II.

[0133] 3. Two consecutive DEAE-cellulose treatments

[0134] The intermediate II was made into a 2% solution, dissolved at 100 °C, cooled to 80 °C, then DEAE-cellulose was added (the dosage of the first DEAE-cellulose treatment was 7 times the mass of the crude agarose, and the dosage of the second treatment was 5 times the mass of the crude agarose), and stirred at 80 °C for 1.5 h. The precipitate was removed by centrifugation, the supernatant was gelled at room temperature and then dried, and finally the obtained dry gel was crushed to obtain intermediate III.

[0135] 4. Preparation of medium and high molecular weight agarose

[0136] At 100 °C, the intermediate III was dissolved in ultrapure water and continuously stirred for 3 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into blocks of about 1 cm x 1 cm, 0.2 M HCl solution was added to the hydrogel blocks, and treated at 37 °C for 10 h to obtain a medium and high molecular weight agarose hydrogel. The obtained medium and high molecular weight agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, then freeze-dried and crushed to obtain medium and high molecular weight agarose powder.

[0137] Comparative Example 2

[0138] 1. Two consecutive DMSO treatments

[0139] The crude agarose obtained in Step 1 of Example 1 was mixed with DMSO (crude agarose:DMSO = 1 g:50 mL) at 75 °C, stirred in an oil bath for 8 h, the precipitate was removed by centrifugation, the supernatant was gelled at room temperature and then dried, and the obtained dry gel was crushed to obtain intermediate I.

[0140] 2. Two consecutive sodium iodide treatments

[0141] The intermediate I was added to the sodium iodide solution at 55 °C (the concentration of the first sodium iodide treatment was 6 g / L, and the concentration of the second sodium iodide treatment was 4 g / L), stirred for 4 h, then filtered by suction and the residual sodium iodide was washed away, and dried to obtain intermediate II.

[0142] 3. Two consecutive DEAE-cellulose treatments

[0143] Prepare Intermediate II into a 2% solution, dissolve it at 100 °C, cool it to 80 °C, add DEAE-cellulose (the dosage of DEAE-cellulose for the first treatment is 7 times the mass of the crude agarose, and the dosage for the second treatment is 5 times the mass of the crude agarose), and stir at 80 °C for 1.5 h. Centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and finally crush the obtained dry gel to obtain Intermediate III.

[0144] 4. Preparation of Medium and High Molecular Weight Agarose

[0145] Dissolve Intermediate III in ultrapure water at 100 °C and continuously stir for 3 min. Then, let the solution stand at 37 °C for 8 h to form an agarose hydrogel. Cut the obtained hydrogel into blocks of about 1 cm x 1 cm, add 0.2 M HCl solution to the hydrogel blocks, and treat at 37 °C for 10 h to obtain a medium and high molecular weight agarose hydrogel. Immerse the obtained medium and high molecular weight agarose hydrogel in ultrapure water, rinse until the pH value of the washing solution is neutral, then freeze-dry and crush to obtain medium and high molecular weight agarose powder.

[0146] Comparative Example 3

[0147] 1. Two consecutive EDTA-Na2 treatments

[0148] Add the crude agarose obtained in Step 1 of Example 1 to an EDTA-Na2 solution (the concentration of the first EDTA-Na2 treatment is 6 g / L, and the concentration of the second EDTA-Na2 treatment is 4 g / L) at 55 °C and stir for 4 h. Then, filter by suction and wash away the residual EDTA-Na2, and dry to obtain Intermediate I.

[0149] 2. Two consecutive DMSO treatments

[0150] Mix Intermediate I with DMSO (crude agarose:DMSO = 1 g:50 mL) at 75 °C, stir in an oil bath for 8 h, centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and crush the obtained dry gel to obtain Intermediate II.

[0151] 3. Two consecutive DEAE-cellulose treatments

[0152] Prepare Intermediate II into a 2% solution, dissolve it at 100 °C, cool it to 80 °C, add DEAE-cellulose (the dosage of DEAE-cellulose for the first treatment is 7 times the mass of the crude agarose, and the dosage for the second treatment is 5 times the mass of the crude agarose), and stir at 80 °C for 1.5 h. Centrifuge to remove the precipitate, dry the supernatant after gelling at room temperature, and finally crush the obtained dry gel to obtain Intermediate III.

[0153] 4. Preparation of Medium and High Molecular Weight Agarose

[0154] At 100 °C, Intermediate III was dissolved in ultrapure water and continuously stirred for 3 min. Then, the solution was allowed to stand at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into pieces of about 1 cm x 1 cm. 0.2 M HCl solution was added to the hydrogel pieces and treated at 37 °C for 10 h to obtain a medium and high molecular weight agarose hydrogel. The obtained medium and high molecular weight agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, and then freeze-dried and pulverized to obtain medium and high molecular weight agarose powder.

[0155] Comparative Example 4

[0156] 1. Two consecutive DMSO treatments

[0157] The crude agarose obtained in Step 1 of Example 1 was mixed with DMSO (crude agarose: DMSO = 1 g: 50 mL) at 75 °C, stirred in an oil bath for 8 h, the precipitate was removed by centrifugation, the supernatant was gelled at room temperature and then dried, and the obtained dry gel was pulverized to obtain Intermediate I.

[0158] 2. Two consecutive DEAE-cellulose treatments

[0159] Intermediate I was prepared into a 2% solution, dissolved at 100 °C, cooled to 80 °C, then DEAE-cellulose was added (the dosage for the first DEAE-cellulose treatment was 7 times the mass of the crude agarose, and the dosage for the second treatment was 5 times the mass of the crude agarose), and stirred at 80 °C for 1.5 h. The precipitate was removed by centrifugation, the supernatant was gelled at room temperature and then dried, and finally the obtained dry gel was pulverized to obtain Intermediate II.

[0160] 3. Two consecutive EDTA-Na2 treatments

[0161] Intermediate II was added to an EDTA-Na2 solution (the concentration for the first EDTA-Na2 treatment was 6 g / L, and the concentration for the second EDTA-Na2 treatment was 4 g / L) at 55 °C and stirred for 4 h. Subsequently, it was filtered and the residual EDTA-Na2 was washed away, and dried to obtain Intermediate III.

[0162] 4. Preparation of Medium and High Molecular Weight Agarose

[0163] At 100 °C, intermediate III was dissolved in ultrapure water and stirred continuously for 3 min. Then, the solution was left standing at 37 °C for 8 h to form an agarose hydrogel. The obtained hydrogel was cut into blocks of about 1 cm x 1 cm, and 0.2 M HCl solution was added to the hydrogel blocks and treated at 37 °C for 10 h to obtain a medium- and high-molecular-weight agarose hydrogel. The obtained medium- and high-molecular-weight agarose hydrogel was soaked in ultrapure water, rinsed until the pH value of the washing solution was neutral, and then freeze-dried and pulverized to obtain medium- and high-molecular-weight agarose powder.

[0164] Table 1: Detection results of agarose prepared in Example 1 and Comparative Examples 1-4

[0165]

[0166] The contents of other impurities in the medium- and high-molecular-weight medical-grade agarose of Example 1 of the present invention were detected by using the existing technology detection method, and the results are shown in Table 2.

[0167] Table 2: Contents of other impurities in the medium- and high-molecular-weight medical-grade agarose prepared in Example 1

[0168]

[0169] As can be seen from Table 1, compared with Comparative Examples 1-4, the content of sulfate radicals in Example 1 was lower, indicating that the medical-grade agarose prepared by the method of the present invention has high purity, low impurity content, and high safety. And, through the detection of the inventor, the medical-grade agarose prepared in Examples 2-4 also has extremely low impurity content (including extremely low sulfate radical content and extremely low impurity content shown in Table 2).

[0170] Physical and chemical property tests of medical-grade agarose with different molecular weights in Example 1

[0171] 1. Physical object shooting of medical-grade agarose with different molecular weights

[0172] 30 mg of high-molecular-weight, medium- and high-molecular-weight, medium-molecular-weight, and low-molecular-weight medical-grade agarose prepared in Example 1 were respectively dissolved in 2 mL of ultrapure water at 100 °C. Subsequently, 1 mL of it was transferred to a transparent vial while it was hot and left standing at room temperature; another 1 mL was placed in the lid of a centrifuge tube and also left standing at room temperature. The physical objects of medical-grade agarose with different molecular weights are as Figure 1 shown. High-molecular-weight, medium- and high-molecular-weight, and medium-molecular-weight medical-grade agarose can all form hydrogels, while low-molecular-weight medical-grade agarose cannot form a gel structure due to its shorter molecular chains and shows liquid fluidity.

[0173] 2. Infrared characterization of medical-grade agarose with different molecular weights

[0174] The Fourier transform infrared spectra of high molecular weight, medium-high molecular weight, medium molecular weight, and low molecular weight medical-grade agarose powders prepared in Example 1 are as follows Figure 2 shown. High molecular weight, medium-high molecular weight, medium molecular weight, and low molecular weight medical-grade agarose all show typical agarose peaks at 3430 cm -1 , 2899 cm -1 , 1073 cm -1 , 930 cm -1 and 890 cm -1 (±5 cm -1 ). After the molecular chain of agarose is shortened, the infrared spectrum remains unchanged, indicating that the basic disaccharide structure and functional groups remain the same, and the infrared spectrum still reflects the complete molecular structure characteristics.

[0175] 3. Gel strength test of medical-grade agarose with different molecular weights

[0176] The high molecular weight, medium-high molecular weight, medium molecular weight, and low molecular weight medical-grade agarose powders prepared in Example 1 were respectively formulated into 1.5% solutions, dissolved at 100 °C for 30 min, cooled, and left standing overnight at room temperature. Then, they were made into gel blocks with a uniform thickness of 1 cm×1 cm×2 mm, and this gel block was placed on the sample stage of a rheometer. The shear strain (1%) of the rheometer was fixed, and G'(Pa) and G"(Pa) of the gel were measured as the angular frequency changed. The test results of the rheological mechanical properties of high molecular weight, medium-high molecular weight, medium molecular weight, and low molecular weight medical-grade agarose hydrogels are as follows Figure 3 shown. The gel strength is in the order of: high molecular weight medical-grade agarose > medium-high molecular weight medical-grade agarose > medium molecular weight medical-grade agarose > low molecular weight medical-grade agarose. The higher the molecular weight, the greater the gel strength.

[0177] 4. Shear viscosity test of medical-grade agarose with different molecular weights

[0178] The high molecular weight, medium-high molecular weight, medium molecular weight, and low molecular weight medical-grade agarose powders prepared in Example 1 were respectively formulated into 1.5% solutions, dissolved at 100 °C for 30 min, cooled, and left standing overnight at room temperature. Then, they were made into gel blocks with a uniform thickness of 1 cm×1 cm×2 mm, and this gel block was placed on the sample stage of a rheometer. A creep scan was performed at a shear rate of 0.01 s -1 ~100 s -1 to obtain the shear viscosity of the gel. The test results of the shear viscosity of high molecular weight, medium-high molecular weight, medium molecular weight, and low molecular weight medical-grade agarose hydrogels are as follows Figure 4As shown in the figure. The shear viscosities are in the order of: high molecular weight medical grade agarose > medium-high molecular weight medical grade agarose > medium molecular weight medical grade agarose > low molecular weight medical grade agarose, that is, the higher the molecular weight, the greater the shear viscosity.

[0179] 5. Injection performance test of medical grade agarose with different molecular weights

[0180] The high molecular weight, medium-high molecular weight, medium molecular weight and low molecular weight medical grade agarose powders prepared in Example 1 were respectively formulated into 1.5% solutions, dissolved at 100 °C for 30 min, and then filled into 1 mL syringes while still hot, cooled overnight at room temperature, and then the needle passing performance of the samples was tested using a needle passing instrument. During the test, the syringe needle was installed, the plunger was pushed at a constant speed, and the sample in the syringe was extruded through the needle to obtain the extrusion force curve.

[0181] The test results of the injection performance of high molecular weight, medium-high molecular weight, medium molecular weight and low molecular weight medical grade agarose hydrogels are as Figure 5 shown. The extrusion forces are in the order of: high molecular weight medical grade agarose > medium-high molecular weight medical grade agarose > medium molecular weight medical grade agarose > low molecular weight medical grade agarose. As the molecular weight decreases, the extrusion force gradually decreases, and at the same time, the extrusion force curve shows a more gentle trend.

[0182] 6. Degradability test of medical grade agarose with different molecular weights

[0183] Appropriate amounts (W0) of freeze-dried sponges of high molecular weight, medium-high molecular weight, medium molecular weight and low molecular weight medical grade agarose were respectively weighed and placed in centrifuge tubes, and immersed in 1.0 M hydrochloric acid solution at 37 °C to characterize the degradation behavior of the hydrogel. Samples were taken at regular intervals, the supernatant was removed after centrifugation, and the precipitate was freeze-dried and weighed (W t ). The acid degradation rate was calculated according to the following formula: Weight loss (%) = (W0 - W t ) / W0 × 100%. The degradation results are shown in Figure 6 .

[0184] The results showed that at day 6, the degradation rate of high molecular weight medical grade agarose was 26%, the degradation rate of medium-high molecular weight medical grade agarose was 53%, the degradation rate of medium molecular weight medical grade agarose was 88%, and the low molecular weight medical grade agarose was completely degraded with a degradation rate of 100%. The overall trend indicates that the lower the molecular weight, the faster the degradation rate.

[0185] 7. Antioxidant capacity test of medical grade agarose with different molecular weights

[0186] The ABTS method was used to evaluate the in vitro antioxidant capacity of agarose. The stable ABTS radical solution is blue-green and has a maximum absorption peak at 734 nm. When the radicals are scavenged by the sample, the number of radicals decreases, the color of the solution gradually fades, and the absorbance at 734 nm decreases accordingly. Therefore, the ability of the sample to scavenge ABTS radicals can be judged by measuring the change in absorbance.

[0187] The experimental procedure is as follows: Prepare a 7.4 mmol / L ABTS stock solution and a 2.6 mmol / L potassium persulfate (K2S2O8) solution, mix them in a 1:1 ratio, and let them stand in the dark for 12 h to generate the ABTS radical solution. Before the experiment, dilute this solution with ultrapure water to an absorbance of 0.70 ± 0.02 and use it as the working solution for standby. After preparing the sample solutions at different concentrations, measure them in a 96-well plate. Add 20 µL of the sample solution and 180 µL of the working solution to each well, react at room temperature in the dark for 3 - 5 min, and then measure the absorbance at 734 nm using a microplate reader. Calculate the scavenging rate according to the following formula: Scavenging rate (%) = [(A0 - A) / A0] × 100, where A0 is the absorbance of the working solution and A is the absorbance after mixing the sample and the working solution. The test results are as Figure 7 shown.

[0188] The results showed that the antioxidant capacities were in the order of: high molecular weight medical grade agarose < medium-high molecular weight medical grade agarose < medium molecular weight medical grade agarose < low molecular weight medical grade agarose. The overall trend indicated that the lower the molecular weight, the stronger the antioxidant capacity.

[0189] 8. Moisturizing effect test of medical grade agarose with different molecular weights on zebrafish

[0190] The experiment studied the change of skin water loss in zebrafish by treating them with sodium chloride. Due to osmotic pressure, water loss on the skin surface leads to shrinkage, and the tail area decreases accordingly. This was used as a model to evaluate the moisturizing effect of the sample. The tested zebrafish were divided into three groups: a normal control group, a model control group, and a test article group. The normal control group was not treated with anything; the model control group was only treated with a sodium chloride solution; the test article group was quantitatively added with the test sample while being treated with sodium chloride (the test sample contacted the zebrafish skin by being leached into the standard dilution water). After incubation for a period of time, the zebrafish were photographed, and the moisturizing effect of the test article was evaluated based on the tail area and tail length of the zebrafish.

[0191] The test samples included: high molecular weight medical grade agarose, medium-high molecular weight medical grade agarose, medium molecular weight medical grade agarose, low molecular weight medical grade agarose, high molecular weight hyaluronic acid, medium molecular weight hyaluronic acid, and low molecular weight hyaluronic acid. The test results are as Figure 8As shown in the figure. The results showed that compared with the model control group, high-molecular-weight medical-grade agarose, medium-high-molecular-weight medical-grade agarose, medium-molecular-weight medical-grade agarose, and low-molecular-weight medical-grade agarose could all significantly promote the recovery of the shrunken tails of zebrafish, making them close to the normal level, and exhibited excellent moisturizing properties comparable to those of hyaluronic acid with different molecular weights.

[0192] 9. Test on the repair effect of medical-grade agarose with different molecular weights on zebrafish

[0193] The repair effects of medical-grade agarose with different molecular weights were evaluated through the regeneration of the caudal fins of zebrafish after caudal fin amputation. Zebrafish embryos were incubated for 72 h, and each fish was anesthetized for about 3 s, quickly placed on a glass slide, and the caudal fin was cut off under a microscope according to the same standard, and then quickly returned to the embryo culture solution. The zebrafish after caudal fin amputation were randomly divided into a normal control group, a model control group, and a test article group, and the zebrafish in each group were placed in a 96-well plate, with 1 fish in each well. At 24 h, 48 h, and 72 h after caudal fin amputation, the fish in each group were taken out respectively, anesthetized, and the regeneration of the caudal fin was observed under a microscope and photographed, and then the regeneration area of the caudal fin of each fish was measured using ImageJ software.

[0194] The test articles included: high-molecular-weight medical-grade agarose, medium-high-molecular-weight medical-grade agarose, medium-molecular-weight medical-grade agarose, and low-molecular-weight medical-grade agarose, high-molecular-weight hyaluronic acid, medium-molecular-weight hyaluronic acid, and low-molecular-weight hyaluronic acid. The test results are as Figure 9 shown. The results showed that compared with the model control group, high-molecular-weight medical-grade agarose, medium-high-molecular-weight medical-grade agarose, medium-molecular-weight medical-grade agarose, and low-molecular-weight medical-grade agarose could all promote the healing and repair of the damaged caudal fins of zebrafish. In addition, agarose degrades slowly, which helps to maintain the stability of the local microenvironment and prolong the repair effect, making the repair effects of medium-molecular-weight and low-molecular-weight medical-grade agarose better than those of hyaluronic acid with the corresponding molecular weights.

[0195] In summary, the present invention provides a method for preparing medical-grade agarose with different molecular weights. The method of the present invention uses a crude agarose product with a molecular weight > 100,000 as a raw material, which is treated with DMSO, EDTA-Na2, and DEAE-cellulose and then treated with hydrochloric acid. By strictly controlling the conditions in the DMSO, EDTA-Na2, and DEAE-cellulose treatment stages and adjusting the concentration, temperature, time, etc. in the hydrochloric acid treatment stage, medical-grade agarose with medium-high molecular weights in the molecular weight range of 30,000 to 100,000, medical-grade agarose with medium molecular weights in the molecular weight range of 3,000 to 30,000, and low molecular weight medical-grade agarose with a molecular weight less than 3,000 are obtained. The lower the molecular weight of the medical-grade agarose prepared by this method, the lower its gel strength and viscosity, the faster the degradation rate, and at the same time, it has better injectability and antioxidant ability. The agarose with different molecular weights prepared by this method all exhibit excellent moisturizing effects and repair functions, and have significant application potential in the fields of medical skin care products and functional dressings.

[0196] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. A method for preparing medical grade agarose with different molecular weights, characterized in that: The following steps are involved: The crude agarose was mixed with dimethyl sulfoxide, heated, centrifuged, and the supernatant was collected to obtain intermediate I; The intermediate I is mixed with EDTA-Na2 solution, heated, filtered, and the filter cake is obtained to obtain the intermediate II; the intermediate II is prepared into a solution, DEAE-cellulose is added, heated, centrifuged, and the supernatant is obtained to obtain the intermediate III; the intermediate III is prepared into an agarose hydrogel and treated with hydrochloric acid to obtain medical grade agarose with different molecular weights.

2. The method for preparing medical grade agarose with different molecular weights according to claim 1, characterized in that: The following steps are involved: (1) Mix the crude agarose with dimethyl sulfoxide, heat at 65-85°C, centrifuge, gel the supernatant and dry to obtain intermediate I; (2) mixing the intermediate I obtained in step (1) with an EDTA-Na2 solution, heating the mixture at 45-95°C, filtering the mixture, and drying the filter cake to obtain an intermediate II; (3) preparing a solution of the intermediate II obtained in step (2), adding DEAE-cellulose, heating at 60-99°C, centrifuging, and gelling the supernatant and drying to obtain the intermediate III; (4) The intermediate III obtained in step (3) is formulated into an agarose hydrogel, and treated with a 0.2 M to 1.5 M hydrochloric acid solution at a temperature of 25 to 90 °C for 2 to 10 h to obtain medical grade agarose with different molecular weights.

3. The method for preparing medical grade agarose with different molecular weights according to claim 2, characterized in that: The mass volume ratio of the crude agarose to dimethyl sulfoxide in step (1) is 1: (40-60) g / mL.

4. The method for preparing medical grade agarose with different molecular weights according to claim 2, characterized in that: The mass fraction of the EDTA-Na2 solution in step (2) is 2-8 g / L.

5. The method for preparing medical grade agarose with different molecular weights according to claim 2, characterized in that: The mass of DEAE-cellulose in step (3) is 1 to 7 times the mass of crude agarose.

6. The method for preparing medical grade agarose with different molecular weights according to claim 2, characterized in that: In step (4), when the concentration of the hydrochloric acid is 0.2 M to 1.5 M, the treatment temperature is 25 to 40 °C, and the treatment time is 8 to 10 h, a medium-high molecular weight medical grade agarose with a molecular weight of 30,000 to 100,000 is obtained; When the concentration of the hydrochloric acid is 0.4 M to 1.5 M, the treatment temperature is 55 to 65 ° C, and the time is 7 to 9 h, a medium molecular weight medical grade agarose with a molecular weight of 3000 to 30,000 is obtained; When the concentration of the hydrochloric acid is 1.2 M to 1.5 M, the treatment temperature is 70 to 90 ° C, and the time is 2 to 6 h, a low molecular weight medical grade agarose with a molecular weight of less than 3000 is obtained.

7. Medical grade agarose of different molecular weights prepared by the method as described in any one of claims 1 to 6.

8. The use of medical grade agarose with different molecular weights as claimed in claim 7 in the biomedical field, characterized in that: include: Use medium-high molecular weight and medium molecular weight medical grade agarose for implants and gel dressings; Use low molecular weight medical grade agarose in anti-aging skin care and skin repair products.

9. A comprehensive preparation suitable for dermatology, characterized in that: The preparation comprises medical-grade agarose of different molecular weights as described in claim 7, wherein the proportion of medical-grade agarose of different molecular weights is adjusted according to actual needs to achieve the synergistic effects of moisturizing, anti-oxidation and repair functions.

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