A multi-component hydrogel containing a neptunea biological active peptide, and a preparation method and application thereof

Abstract

The application discloses a kind of multi-component hydrogel comprising active peptide of Neptune and its preparation method and application, it is related to biological gel and active peptide technical field;Including the mass concentration of 10% oxidized carboxymethyl cellulose aqueous solution, the mass concentration of 5% carboxymethyl chitosan aqueous solution and the mass concentration of 0.1% active peptide of Neptune aqueous solution;The oxidized carboxymethyl cellulose aqueous solution and carboxymethyl chitosan aqueous solution are obtained by preparation from PBS buffer solution of pH=7.4;Multi-component hydrogel comprising active peptide of Neptune of the application has good mechanical properties and biocompatibility, can adhere to the surface of wound mouth, play the role of isolation pathogenic microorganism and human tissue cell, can promote cell migration, promote wound healing, show excellent antibacterial performance to bacteria, fungi and some drug-resistant bacteria.

Description

Technical Field

[0001] This invention relates to the field of biogels and bioactive peptides, specifically a multi-component hydrogel containing spirulina bioactive peptides, its preparation method, and its applications. Background Technology

[0002] The skin is the body's first line of defense, protecting against external pathogens. However, the skin is often damaged for various reasons. When a healthy individual is injured, the body can restore the normal structure and function of the skin through self-repair. However, this self-repair ability has limited effectiveness in repairing severe injuries and chronic wounds caused by deep burns, diabetes, trauma, etc.

[0003] Existing medical hydrogel antibacterial materials are generally designed with a thin film adsorbed on the surface to isolate pathogenic microorganisms from human tissue cells, exhibiting significant antibacterial and bacteriostatic effects against pathogenic microorganisms, while also accelerating wound healing and preventing penetration. However, medical hydrogel antibacterial materials generally suffer from poor biocompatibility and slow wound healing during use. Furthermore, prolonged use can lead to drug resistance in bacteria, fungi, and even drug-resistant strains, affecting wound healing speed and antibacterial efficacy.

[0004] Therefore, there is an urgent need to develop bioactive medical hydrogels that can ensure both biocompatibility and bactericidal properties while also promoting wound healing. Summary of the Invention

[0005] To address the issues of poor biocompatibility, bactericidal properties, and poor wound healing of hydrogels, the present invention aims to provide a multi-component hydrogel containing spirulina bioactive peptides, its preparation method, and its applications.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0008] Preferably, the volume ratio of the oxidized carboxymethyl cellulose aqueous solution to the carboxymethyl chitosan aqueous solution is 3-7:3-7; and the volume ratio of the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution to the spirulina bioactive peptide aqueous solution is 10-15:1.

[0009] Preferably, it further includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of the aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 5 to 8:100.

[0010] Preferably, the oxidized carboxymethyl cellulose is prepared according to the following steps:

[0011] Sodium periodate was dissolved in water, and the pH was adjusted to 4.8-5.2. Carboxymethyl cellulose was added and mixed evenly. The mixture was stirred at 30-35°C in the dark for 4-6 hours. Ethanol was added and stirred for 1-2 hours to obtain a reaction solution. The mixture was poured into the first methanol and stirred for 10-30 minutes. The mixture was filtered, and the solid was washed with the second methanol and dried to obtain oxidized carboxymethyl cellulose.

[0012] The mass ratio of sodium periodate, water, carboxymethyl cellulose, ethanol, first methanol, and second methanol is 6-7:150:5-5.5:100-120:450-550:50-80.

[0013] Preferably, the degree of oxidation of the oxidized carboxymethyl cellulose is 35% to 40%.

[0014] Preferably, the degree of carboxylation of the carboxymethyl chitosan is ≥80%.

[0015] Preferably, the degree of substitution of carboxymethyl groups in the sodium carboxymethyl β-glucan is 60-70%.

[0016] This invention also includes a method for preparing a multi-component hydrogel containing snail bioactive peptides, comprising the following steps:

[0017] Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use;

[0018] Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution with pH=7.4, and set aside for later use;

[0019] Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides for later use;

[0020] A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides were mixed in a volume ratio and stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptides.

[0021] This invention also includes a method for preparing a multi-component hydrogel containing snail bioactive peptides, comprising the following steps:

[0022] Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use;

[0023] Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution with pH=7.4, and set aside for later use;

[0024] Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides for later use;

[0025] A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides were mixed in a volume ratio to obtain a mixed solution, which was then weighed and set aside for later use.

[0026] Sodium carboxymethyl β-glucan was added to the mixture and stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptides;

[0027] The mass ratio of sodium carboxymethyl β-glucan to the mass of the mixture is 5-8:100.

[0028] The present invention also includes the application of multi-component hydrogels containing spirulina bioactive peptides for wound repair.

[0029] The present invention has the following advantages over the prior art:

[0030] The multi-component hydrogel containing spirulina bioactive peptides of the present invention has good mechanical properties and biocompatibility. It can adhere to the surface of wounds, isolate pathogenic microorganisms from human tissue cells, promote cell migration, promote wound healing, and exhibit excellent antibacterial properties against bacteria, fungi, and some drug-resistant bacteria.

[0031] This invention relates to a multi-component hydrogel containing snail bioactive peptides, including snail bioactive peptides extracted from marine snails. These peptides exhibit good in vivo and in vitro antioxidant activity, effectively scavenging free radicals and demonstrating excellent antibacterial properties against bacteria, fungi, and certain drug-resistant pathogens. This invention prepares the hydrogel by mixing snail bioactive peptides, carboxymethyl chitosan, and oxidized carboxymethyl cellulose. On one hand, the amino groups in carboxymethyl chitosan can form Schiff bases with the aldehyde groups in oxidized carboxymethyl cellulose, thereby cross-linking to form a gel.

[0032] In the preferred formulation, the sodium carboxymethyl β-glucan salt in sodium carboxymethyl β-glucan can also participate in gel formation through ionic and hydrogen bonds, endowing the gel with good mechanical properties. Adding spirochete bioactive peptides to the hydrogel imparts good antibacterial, antioxidant, and cell migration-promoting properties, which are beneficial for wound healing. Sodium carboxymethyl β-glucan, derived from yeast, has a hyperbranched structure and can bind to many immune-related receptors in host cells, thus regulating local immune function and accelerating wound healing. Attached Figure Description

[0033] Figure 1 The following are the rheological property test results of the hydrogels of the present invention, wherein (A) is the shear thinning test of different hydrogels, (B) is the modulus-angular frequency curve of different hydrogels, (C) is the strain scan of HY hydrogel in the range of 0.1%-100%, (D) is the five rheological cycles of HY hydrogel around the critical point, and (E) is the modulus-angular frequency curve of HYP hydrogel.

[0034] Figure 2 The results demonstrate the injectability and self-healing properties of the hydrogel;

[0035] Figure 3 Cell viability of L929 cells after incubation with hydrogel extract of bioactive peptides from snails for 24 h, 48 h, and 72 h. Figure 4 Live (Calcein-AM) / dead (PI) staining of L929 cells after incubation with hydrogel extract of spirulina bioactive peptides for 24 h, 48 h, and 72 h (scale bar, 50 μm). The images are composed of the green channel (Calcein / AM) and the red channel (PI).

[0036] Figure 5 The results are from the hemolysis experiment of the hydrogel;

[0037] Figure 6 (A) Representative images of L929 cells after incubation with 200 μg / mL HY and HYP hydrogel extracts for 24 h (scale bar, 50 μm) (B) Migration rate of L929 cells at different times;

[0038] Figure 7 To assess the antibacterial activity of hydrogels: (A) Photograph of surviving bacteria on an agar plate; (B) Quantitative analysis of the antibacterial activity of hydrogels against E. coli; (C) Quantitative analysis of the antibacterial activity of hydrogels against S. aureus.

[0039] Figure 8The antioxidant capacity of the hydrogels (A) Color changes of each group after 30 min of incubation (B) Scavenging rate of DPPH free radicals of different groups (C) UV-Vis spectra of each group after 30 min of incubation;

[0040] Figure 9 To evaluate the wound healing activity of the hydrogel in a rat model. Detailed Implementation

[0041] The purpose of this invention is to provide a multi-component hydrogel containing spirulina bioactive peptides, its preparation method, and its applications, which is achieved through the following technical solutions:

[0042] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0043] Preferably, the volume ratio of the oxidized carboxymethyl cellulose aqueous solution to the carboxymethyl chitosan aqueous solution is 3-7:3-7; and the volume ratio of the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution to the spirulina bioactive peptide aqueous solution is 10-15:1.

[0044] Preferably, it further includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of the aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 5 to 8:100.

[0045] Preferably, the oxidized carboxymethyl cellulose is prepared according to the following steps:

[0046] Sodium periodate was dissolved in water, and the pH was adjusted to 4.8-5.2. Carboxymethyl cellulose was added and mixed evenly. The mixture was stirred at 30-35°C in the dark for 4-6 hours. Ethanol was added and stirred for 1-2 hours to obtain a reaction solution. The mixture was poured into the first methanol and stirred for 10-30 minutes. The mixture was filtered, and the solid was washed with the second methanol and dried to obtain oxidized carboxymethyl cellulose.

[0047] The mass ratio of sodium periodate, water, carboxymethyl cellulose, ethanol, first methanol, and second methanol is 6-7:150:5-5.5:100-120:450-550:50-80.

[0048] Preferably, the degree of oxidation of the oxidized carboxymethyl cellulose is 35% to 40%.

[0049] Preferably, the degree of carboxylation of the carboxymethyl chitosan is ≥80%.

[0050] Preferably, the degree of substitution of carboxymethyl groups in the sodium carboxymethyl β-glucan is 60-70%.

[0051] This invention also includes a method for preparing a multi-component hydrogel containing snail bioactive peptides, comprising the following steps:

[0052] Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use;

[0053] Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution with pH=7.4, and set aside for later use;

[0054] Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides for later use;

[0055] A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides were mixed in a volume ratio and stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptides.

[0056] This invention also includes a method for preparing a multi-component hydrogel containing snail bioactive peptides, comprising the following steps:

[0057] Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use;

[0058] Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution with pH=7.4, and set aside for later use;

[0059] Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides for later use;

[0060] A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides were mixed in a volume ratio to obtain a mixed solution, which was then weighed and set aside for later use.

[0061] Sodium carboxymethyl β-glucan was added to the mixture and stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptides;

[0062] The mass ratio of sodium carboxymethyl β-glucan to the mass of the mixture is 5-8:100.

[0063] The present invention also includes the application of multi-component hydrogels containing spirulina bioactive peptides for wound repair.

[0064] The raw material used in this invention, snail bioactive peptide, was obtained according to the method described in application number 201810916171.7.

[0065] Rheological property evaluation

[0066] During the research and development process, the rheological properties of the hydrogel were explored by adjusting the volume ratio of a 10% (w / w) oxidized carboxymethyl cellulose (CMC) aqueous solution and a 5% (w / w) carboxymethyl chitosan aqueous solution (CMCS). The rheological properties of the hydrogel were measured using a rotational rheometer. To characterize the injectability of the hydrogel, the temperature was set at 25℃, the frequency scanning mode was used, and the shear rate ranged from 0.1 to 100 s⁻¹. -1 Viscosity analysis was performed on different hydrogels under the specified conditions. To characterize the gelling properties of the hydrogels, the viscosity was analyzed at a set temperature of 37℃, a strain of 1%, and an angular frequency of 0.1-100 rad / s. -1 In frequency scanning mode, the storage modulus (G′) and loss modulus (G″) of different hydrogels were analyzed. To determine the critical strain point of the hydrogel, the changes in G′ and G″ were investigated in amplitude scanning mode at 25℃ and strain γ = 0.1-100%. To characterize the self-healing property of the hydrogel, alternating strain scanning was performed at 25℃ and 1Hz, and the test was repeated for 5 cycles to examine the changes in G′ and G″.

[0067] The rheological property test results are as follows Figure 1 As shown. The viscosity of the gel has a significant impact on injectability; excessively high viscosity is detrimental to hydrogel injection. From Figure 1 As shown in -A, the viscosity of different hydrogels decreases with increasing shear rate, indicating that the prepared hydrogels have shear-thinning properties, which is beneficial for injectability. However, the viscosities of different hydrogels vary. The hydrogels CMCS / CMC 4 / 6 and CMCS / CMC 7 / 3 have higher viscosities at low shear rates, requiring higher shear rates to achieve lower viscosities. The hydrogel CMCS / CMC 5 / 5 achieves lower viscosities at lower shear rates, showing better performance.

[0068] The modulus of the hydrogel affects its gelling properties. If G′ is higher than the corresponding G″, the hydrogel has good gelling properties and a stable structure. Conversely, if G′ is lower than G″, it indicates that the hydrogel has poor internal cross-linking and is easily broken. Figure 1-B indicates that the storage modulus (G′) of all five prepared hydrogels was higher than the loss modulus (G”), indicating that the hydrogels had good gelling properties. However, when only CMCS and CMC were added, the storage modulus (G′) and loss modulus (G”) of the CMCS / CMC 3 / 7 hydrogel were relatively low, resulting in poor mechanical properties. Therefore, sodium carboxymethyl β-glucan should be added to CMCS / CMC 3 / 7 when using it. The CMCS / CMC 5 / 5 hydrogel had a more suitable storage modulus (G′) and loss modulus (G”), showing even better performance. Therefore, we selected the CMCS / CMC 5 / 5 hydrogel as the preferred hydrogel and named it HY. The hydrogel containing spirochete bioactive peptides prepared based on this hydrogel was named HYP and used for subsequent research.

[0069] Determining the critical strain point is beneficial for subsequent research on the self-healing properties of hydrogels. Figure 1 -C indicates that the critical strain value of HY hydrogel is 35%. Preliminary studies on the self-healing properties of HY hydrogel are as follows: Figure 1 As shown in Figure -D, the results indicate that G′ and G″ of the HY hydrogel can still recover to approximately their original values ​​after 5 rheological cycles, exhibiting elastic behavior similar to that of a solid. This suggests that the reconstruction of the internal network of the hydrogel is beneficial to its self-healing. Figure 1 -E shows the modulus-angular frequency curve of the HYP hydrogel. It can be seen that after the addition of spirulina bioactive peptides, the storage modulus (G′) of the hydrogel is still much higher than the loss modulus (G”), which also makes the hydrogel maintain good gelling properties.

[0070] Taking HY as an example, we can further intuitively evaluate the injectability and self-healing properties of hydrogels.

[0071] Injectability: The hydrogel was pre-loaded into a 5mL syringe, and the hydrogel was squeezed out through the needle to write "QLUT" and draw a certain shape to check the injectability of the hydrogel. Self-healing properties: Two hydrogel discs were prepared, one stained with Rhodamine B and the other with Alkali Blue. The two gels were cut into two pieces of equal size, and then the two pieces of gel with different colors were placed in contact for 30 minutes without external force. The self-healing properties of the hydrogels were observed by suspending them.

[0072] The results are as follows Figure 2 As shown, the prepared hydrogel can be extruded through a syringe needle and can smoothly write "QLUT" and draw a heart shape. When circular hydrogels are cut and spliced ​​together, after 30 minutes, the spliced ​​hydrogels form a complete hydrogel capable of supporting its own weight. These results demonstrate that the HY hydrogel prepared in this invention possesses excellent injectability and self-healing properties. Similar results were observed with hydrogels of other proportions.

[0073] The present invention will be further described below with reference to specific embodiments.

[0074] Example 1

[0075] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0076] Example 2

[0077] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0078] The volume ratio of oxidized carboxymethyl cellulose aqueous solution (CMC) to carboxymethyl chitosan aqueous solution (CMCS) is 7:3; the volume ratio of the combined volume of oxidized carboxymethyl cellulose aqueous solution and carboxymethyl chitosan aqueous solution to the volume ratio of spirulina bioactive peptide aqueous solution is 10:1.

[0079] Example 3

[0080] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0081] The volume ratio of oxidized carboxymethyl cellulose aqueous solution (CMC) to carboxymethyl chitosan aqueous solution (CMCS) is 6:4; the volume ratio of the combined volume of oxidized carboxymethyl cellulose aqueous solution and carboxymethyl chitosan aqueous solution to the volume ratio of spirulina bioactive peptide aqueous solution is 15:1.

[0082] The oxidized carboxymethyl cellulose was prepared according to the following steps:

[0083] Sodium periodate was dissolved in water, the pH was adjusted to 4.8, carboxymethyl cellulose was added, and the mixture was stirred evenly. The mixture was stirred at 30°C in the dark for 4 hours, and ethanol was added and stirred for 1 hour to obtain a reaction solution. The mixture was poured into the first methanol and stirred for 10 minutes. The mixture was filtered, the solid was washed with the second methanol, and dried to obtain oxidized carboxymethyl cellulose.

[0084] The mass ratio of sodium periodate, water, carboxymethyl cellulose, ethanol, first methanol, and second methanol is 6:150:5:100:450:50.

[0085] The degree of oxidation of the oxidized carboxymethyl cellulose is 35%;

[0086] The degree of carboxylation of the carboxymethyl chitosan is ≥80%.

[0087] Example 4

[0088] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0089] The volume ratio of oxidized carboxymethyl cellulose aqueous solution (CMC) to carboxymethyl chitosan aqueous solution (CMCS) is 5:5; the volume ratio of the combined volume of oxidized carboxymethyl cellulose aqueous solution and carboxymethyl chitosan aqueous solution to the volume ratio of spirulina bioactive peptide aqueous solution is 11:1.

[0090] The oxidized carboxymethyl cellulose was prepared according to the following steps:

[0091] Sodium periodate was dissolved in water, the pH was adjusted to 5.2, carboxymethyl cellulose was added, and the mixture was stirred evenly at 35°C in the dark for 6 hours. Ethanol was added and stirred for 2 hours to obtain a reaction solution. The mixture was poured into the first methanol and stirred for 30 minutes. The mixture was filtered, the solid was washed with the second methanol, and dried to obtain oxidized carboxymethyl cellulose.

[0092] The mass ratio of sodium periodate, water, carboxymethyl cellulose, ethanol, first methanol, and second methanol is 7:150:5.5:120:550:80.

[0093] The degree of oxidation of the oxidized carboxymethyl cellulose is 40%;

[0094] The degree of carboxylation of the carboxymethyl chitosan is ≥80%.

[0095] Example 5

[0096] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0097] The volume ratio of oxidized carboxymethyl cellulose aqueous solution (CMC) to carboxymethyl chitosan aqueous solution (CMCS) is 4:6; the volume ratio of the combined volume of oxidized carboxymethyl cellulose aqueous solution and carboxymethyl chitosan aqueous solution to the volume ratio of spirulina bioactive peptide aqueous solution is 12:1.

[0098] The oxidized carboxymethyl cellulose was prepared according to the following steps:

[0099] Sodium periodate was dissolved in water, the pH was adjusted to 5, carboxymethyl cellulose was added, and the mixture was stirred evenly. The mixture was stirred at 32°C in the dark for 5 hours, and ethanol was added and stirred for 1.5 hours to obtain a reaction solution. The mixture was poured into the first methanol and stirred for 20 minutes. The mixture was filtered, the solid was washed with the second methanol, and dried to obtain oxidized carboxymethyl cellulose.

[0100] The mass ratio of sodium periodate, water, carboxymethyl cellulose, ethanol, first methanol, and second methanol is 6.5:150:5.2:110:500:70.

[0101] The degree of oxidation of the oxidized carboxymethyl cellulose is 38%;

[0102] The degree of carboxylation of the carboxymethyl chitosan is ≥80%.

[0103] Example 6

[0104] A multi-component hydrogel containing spirulina bioactive peptides comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides; wherein the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution are both prepared from a PBS buffer solution at pH 7.4.

[0105] The volume ratio of oxidized carboxymethyl cellulose aqueous solution (CMC) to carboxymethyl chitosan aqueous solution (CMCS) is 3:7; the volume ratio of the combined volume of oxidized carboxymethyl cellulose aqueous solution and carboxymethyl chitosan aqueous solution to the volume ratio of spirulina bioactive peptide aqueous solution is 12:1.

[0106] Example 7

[0107] A multi-component hydrogel containing spirulina bioactive peptides, compared with Example 2, further includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 5:100; the degree of carboxymethyl substitution in the sodium carboxymethyl β-glucan is 60%.

[0108] Example 8

[0109] A multi-component hydrogel containing spirulina bioactive peptides, compared with Example 3, further includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 8:100; the degree of carboxymethyl substitution in the sodium carboxymethyl β-glucan is 70%.

[0110] Example 9

[0111] A multi-component hydrogel containing spirulina bioactive peptides, compared with Example 4, further includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 6:100; the degree of carboxymethyl substitution in the sodium carboxymethyl β-glucan is 68%.

[0112] Example 10

[0113] A multi-component hydrogel containing spirulina bioactive peptides, compared with Example 5, further includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 7:100; the degree of carboxymethyl substitution in the sodium carboxymethyl β-glucan is 62%.

[0114] Example 11

[0115] A multi-component hydrogel containing spirulina bioactive peptides, compared with Example 6, further includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 8:100; the degree of carboxymethyl substitution in the sodium carboxymethyl β-glucan is 65%.

[0116] The preparation method of the multi-component hydrogel containing spirulina bioactive peptides in Example 2 includes the following steps:

[0117] Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use;

[0118] Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution with pH=7.4, and set aside for later use;

[0119] Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides using deionized water for later use;

[0120] A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose and a 5% (w / w) aqueous solution of carboxymethyl chitosan were mixed at a volume ratio of 7:3, and the sum of the volumes of the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution was mixed with the volume ratio of the spirulina bioactive peptide aqueous solution of 10:1. The mixture was stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptide.

[0121] The preparation methods of Examples 3-6 can be carried out according to the above steps, only the ratio of oxidized carboxymethyl cellulose aqueous solution, carboxymethyl chitosan aqueous solution and spirulina bioactive peptide aqueous solution needs to be changed.

[0122] The preparation method of the multi-component hydrogel containing spirulina bioactive peptides in Example 7 includes the following steps:

[0123] Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use;

[0124] Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution with pH=7.4, and set aside for later use;

[0125] Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides using deionized water for later use;

[0126] A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose and a 5% (w / w) aqueous solution of carboxymethyl chitosan were mixed at a volume ratio of 7:3, and the sum of the volumes of the oxidized carboxymethyl cellulose aqueous solution and the carboxymethyl chitosan aqueous solution was mixed with a 10:1 volume ratio of the spirulina bioactive peptide aqueous solution to obtain a mixed solution. The mixture was weighed and set aside for later use.

[0127] Sodium carboxymethyl β-glucan was added to the mixture and stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptides;

[0128] The mass ratio of sodium carboxymethyl β-glucan to the mass of the mixture is 5:100.

[0129] The preparation methods of Examples 8-11 can be carried out according to the above steps, only by changing the ratio of oxidized carboxymethyl cellulose aqueous solution, carboxymethyl chitosan aqueous solution, spirochete bioactive peptide aqueous solution and carboxymethyl β-glucan sodium.

[0130] 1. Biocompatibility evaluation

[0131] 1.1 Study on the cytotoxicity of hydrogels

[0132] The cytotoxicity of hydrogels to L929 cells was studied using the CCK-8 assay. Lyophilized hydrogels were extracted for 12 hours in MEM complete medium containing 1% penicillin-antibiotic and 10% fetal bovine serum to obtain extracts (50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL). L929 cells were passaged 2-3 times, and cells in the logarithmic growth phase were harvested and cultured at 5 × 10⁶ cells / well. 3 Cells were seeded at a concentration of [number] cells per well in 96-well plates and incubated for 12 hours. Then, the culture medium was removed; the experimental groups were replaced with 200 μL of the corresponding concentration of hydrogel extract, and the control group was replaced with 200 μL of MEM medium. After 24 h, 48 h, and 72 h of culture, the original culture medium was removed, and 100 μL of fresh MEM medium containing 10% CCK-8 reagent was added, followed by incubation for 1–2 hours. Finally, the absorbance was measured at 450 nm using a microplate reader. The cell viability assay formula is as follows:

[0133]

[0134] Where: OD 实验组 OD 对照组 OD 空白组 The absorbance values ​​represent the absorbance values ​​of the experimental group, the control group, and the blank group, respectively.

[0135] The results of the detection of hydrogel cytotoxicity using the CCK-8 kit are as follows: Figure 3 As shown, the results indicate that the relative cell viability in hydrogel extracts of different concentrations of snail bioactive peptides cultured at 24h and 48h was higher than 98%, and the relative cell viability in hydrogel extracts of different concentrations of snail bioactive peptides cultured at 72h was higher than 84%. This demonstrates that the snail bioactive peptide hydrogel prepared by this invention has good biocompatibility, meets relevant regulations (relative cell viability > 80%), and has no obvious toxic effects on normal human cells.

[0136] 1.2 Biocompatibility with cells

[0137] The biocompatibility of the hydrogel with L929 cells was further investigated using live / dead cell staining. The preparation of the hydrogel extract and the cell culture method were the same as those described in the CCK-8 method. After culturing for 24 h, 48 h, and 72 h, 5 μL of Calcein-AM and 5 μL of LPI were added to 5 mL of detection buffer, shaken, and mixed to obtain the live / dead staining working solution. The culture medium was then aspirated from the 96-well plate and washed twice with PBS. Subsequently, 100 μL of the live / dead staining working solution was added to each well, and the plate was incubated in the dark for 30 min. Finally, the cells were photographed using a fluorescence microscope under dark conditions; live cells appeared green, and dead cells appeared red.

[0138] The live / dead cell staining experiment further provided a direct and intuitive analysis of the hydrogel's biocompatibility, and the results are as follows: Figure 4 As shown in Example 4 (using it as an example), the results showed that all experimental groups exhibited similar cell activity to the Control group at 24h, 48h, and 72h, which is consistent with the results of CCK-8 assays. Furthermore, the live cells (green) were spindle-shaped, with only a few dead cells (red), which we speculate is due to apoptosis caused by normal cell metabolism. In addition, the cell density at 48h and 72h was significantly higher than that at 24h. In conclusion, the staining results indicate that the snail bioactive peptide hydrogel prepared in this invention has good biocompatibility.

[0139] 1.3 Blood compatibility studies

[0140] Blood compatibility studies of hydrogels were conducted using sodium citrate-anticoagulated rabbit blood. Lyophilized hydrogel samples were extracted in PBS for 12 hours to obtain a hydrogel extract (5 mg / mL). 1 mL of fresh sodium citrate-anticoagulated rabbit blood was added to 10 mL of PBS, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. The resulting precipitate (red blood cells) was washed three times with PBS. After the third wash, the red blood cell precipitate was diluted in PBS to prepare a 5% red blood cell suspension. Then, 100 μL of the red blood cell suspension was added to 1.1 mL of hydrogel extract, 1.1 mL of PBS, and 1.1 mL of Triton X-100, respectively, and mixed thoroughly to prepare experimental, negative control, and positive control samples. The centrifuge tubes were then incubated at 37°C for 2 hours. After incubation, the tubes were centrifuged at 2000 rpm for 10 minutes, and photographed. 100 μL of the supernatant from each group was transferred to three wells of a 96-well plate, with three parallel wells for each sample. The OD value was measured at λ = 540 nm using a microplate reader. The formula for calculating the hemolysis rate is as follows:

[0141]

[0142] Where: OD 实验组 OD 阴性对照组 OD 阳性对照组 The absorbance values ​​represent the absorbance values ​​of the experimental group, the negative control group, and the positive control group, respectively.

[0143] Hemolysis rate is an important indicator for evaluating the blood compatibility of materials. The principle is that when a material comes into contact with red blood cells, the cells may rupture, leading to the leakage of hemoglobin. The hemolysis rate can be calculated by measuring the absorbance of hemoglobin at a wavelength of 540 nm. A higher hemolysis rate indicates greater damage to red blood cells. The measurement results are as follows: Figure 5As shown in the figure. The results show that the hemolysis rate of the hydrogel HYP containing spirulina bioactive peptides prepared in this invention is 0.74%, and the hemolysis rate of the blank hydrogel HY is 0.41%, both of which are lower than the evaluation standard of 5%, indicating that the prepared hydrogel has good blood compatibility and meets the relevant standards for biomaterials.

[0144] 2. Evaluation of cell migration promotion ability

[0145] The ability of hydrogels to promote cell migration was evaluated using a cell scratch assay. Lyophilized hydrogels were extracted for 12 hours in MEM complete medium containing 1% penicillin-antibiotic and 10% fetal bovine serum to obtain an extract (200 μg / mL). L929 cells were passaged 2-3 times, and cells in the exponential growth phase were collected and cultured at 5 × 10⁶ cells / well. 5 Cells were evenly seeded at a density in 6-well plates and cultured for 12 h. The culture medium was then removed, and cells were streaked vertically using a 200 μL pipette tip. The cells were washed three times with PBS to remove any detached cells. 2 mL of hydrogel extraction buffer was added to each well of the experimental group, and 2 mL of complete culture medium was added to each well of the control group. Changes in the streaked area were observed under a microscope at predetermined time points, and photographs were taken. The streaked area at predetermined time points was measured using Image-J software. Migration rate was calculated using the following formula:

[0146]

[0147] In the formula: S i S0 represents the area of ​​the scratched region at the predetermined time, where S0 is the area of ​​the scratched region at 0h.

[0148] The effects of hydrogel on the proliferation and migration of L929 cells were evaluated using a cell scratch assay to simulate the wound healing process. Results are as follows: Figure 6 As shown, Figure 6 -A represents the cell migration of L929 cells after incubation for 24 hours with the extracts of the 200 μg / mL blank hydrogel control group (HY group) and the multi-component hydrogel containing spirulina bioactive peptides obtained in Example 4 (HYP group). It can be seen that the cell migration area of ​​different groups increased significantly over time. Figure 6 -B shows the migration rate of L929 cells at different time points. It can be seen that both the HYP group and the HY group exhibited higher cell migration rates than the Control group (blank group), with the HYP group showing a higher migration rate than the HY group. At 24 h, the cell migration rates of the HYP group and the HY group were 78.1% and 73.0%, respectively, while the Control group (blank group) was 70.5%. These results indicate that hydrogels containing bioactive peptides can effectively promote the migration of L929 cells.

[0149] 3. Evaluation of antibacterial properties

[0150] The antibacterial activity of hydrogels against Staphylococcus aureus and Escherichia coli was studied using a colony counting method. Lyophilized hydrogels were extracted in PBS for 12 hours to obtain extracts (3 mg / mL, 6 mg / mL, 9 mg / mL, 12 mg / mL). Resuscitated Escherichia coli and Staphylococcus aureus were streaked onto plates, and the culture medium was incubated at 30°C for 24 hours to obtain single colonies. One to two single colonies of Escherichia coli and Staphylococcus aureus were then picked and placed in 10 mL of liquid LB medium, and incubated at 37°C in a shaker for 8 hours. The single colony cultures of Escherichia coli and Staphylococcus aureus were then diluted 50–100 times with PBS. For the experimental group, 100 μL of diluted *Escherichia coli* and *Staphylococcus aureus* were added to 1 mL of hydrogel extracts of different concentrations, followed by 1.9 mL of liquid LB medium, resulting in final concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL, respectively. For the control group, 100 μL of diluted *Escherichia coli* and *Staphylococcus aureus* were added to 2.9 mL of liquid LB medium. Both experimental and control groups were then incubated in shake flasks at 37°C for 12 h. After 12 h of incubation, all groups were diluted 10 μL each time. 5 Take 100 μL of each sample and spread it evenly on agar medium using a spreader. Each group should have at least three replicates. Incubate at 30°C for 24–36 hours, then photograph and record the results, and count the colonies. The inhibition rate is calculated using the following formula:

[0151]

[0152] In the formula: BC c and BC e These represent the colony counts in the control group and the experimental group, respectively.

[0153] The experimental results of antibacterial properties are as follows: Figure 7 As shown. From Figure 7 As can be clearly seen in Figure A, the prepared hydrogel extract exhibits a high inhibition rate against Staphylococcus aureus at 4 mg / mL, significantly higher than its inhibition rate against Escherichia coli. We believe this difference is due to the more complex cell wall structure of Escherichia coli compared to Staphylococcus aureus. This complex cell wall structure may negatively affect the binding of the amino groups in carboxymethyl chitosan and the cationic charges in spirochete bioactive peptides with the negatively charged groups in Escherichia coli, leading to a decrease in the material's inhibitory rate against Gram-negative bacteria. Furthermore, Figure 7 -B and Figure 7Quantitative analysis of the antibacterial activity of the hydrogel extract showed that the antibacterial rates against Escherichia coli and Staphylococcus aureus increased to varying degrees with increasing concentration of the extract. The hydrogel HYP loaded with spirulina bioactive peptides showed an antibacterial rate of 41% against Escherichia coli at 4 mg / mL, higher than the 36% antibacterial rate of the blank control hydrogel HY at 4 mg / mL. The hydrogel HYP loaded with spirulina bioactive peptides also showed an antibacterial rate of up to 95% against Staphylococcus aureus at 4 mg / mL, higher than the 83% antibacterial rate of the blank control hydrogel HY at 4 mg / mL. These results indicate that the addition of spirulina bioactive peptides enhances the antibacterial activity of the hydrogel.

[0154] 4. Evaluation of antioxidant properties

[0155] The antioxidant capacity of the hydrogel was evaluated by measuring its DPPH free radical scavenging efficiency. 0.1 g of lyophilized hydrogel was added to 2 mL of deionized water and extracted for 12 h to obtain the hydrogel extract. For the experimental group, 1 mL of 0.2 mM DPPH methanol solution was added to the above hydrogel extract. For the blank group, only 3 mL of DPPH methanol solution was added. All groups were incubated at room temperature in the dark with stirring for 30 min. After incubation, photographs were taken and recorded. 100 μL of each group was transferred to a 96-well plate, with at least three replicates per group. The absorbance was then measured at 517 nm using a microplate reader. The DPPH scavenging rate was calculated using the following formula:

[0156]

[0157] In the formula, OD 空白组 The absorbance value of the control group (DPPH + methanol) is represented by OD. 实验组 This represents the absorbance value of the experimental group (DPPH + methanol + sample).

[0158] In infections, excessive ROS can lead to cell damage. Enhancing the antioxidant activity of wound dressings is crucial for the wound healing process. The antioxidant results of the prepared hydrogel are as follows: Figure 8 As shown. Figure 8 -A indicates the color changes of each group after 30 minutes of incubation. It can be seen that the hydrogel group is significantly lighter in color compared with the blank group. Figure 8 -B represents the quantitative analysis of the DPPH scavenging rate of different groups. The results show that the hydrogel containing snail bioactive peptides (HYP group) scavenged DPPH free radicals at a rate of 33%, which is higher than the DPPH free radical scavenging rate of the blank control group hydrogel (HY group) at 19%. Figure 8-C shows the UV-Vis spectra of each group after 30 min of incubation. It can be seen that the absorbance values ​​of each hydrogel group near the wavelength of 517 nm are significantly lower than those of the blank group, and the absorbance value of the hydrogel loaded with spirulina bioactive peptides is even lower, indicating that the prepared hydrogel containing spirulina bioactive peptides has good antioxidant capacity.

[0159] 5. Evaluation of activity in promoting wound healing

[0160] A full-thickness skin defect model was established using SD rats (approximately 250g) to investigate the wound-healing activity of hydrogels. Rats were divided into a blank group, a HYP group (spirulina bioactive peptide hydrogel group), and a HY group (blank hydrogel control group). All rats were normally fed for one week, then fasted for 12 hours, anesthetized with 10% chloral hydrate, and their dorsal villi were removed. A 10mm diameter skin wound was created using a 10mm diameter skin punch. 20μL of bacterial suspension (10... 4 CFU / mL of Escherichia coli and Staphylococcus aureus were instilled into the wounds and allowed to act for 2 hours to induce infection. Two hours later, the wounds of rats in the control group were left untreated, while the wounds of the HYP group were covered with spirulina bioactive peptide hydrogel, and the wounds of the HY group were covered with blank hydrogel. All rats were kept in individual cages and fed normally. The wounds of the rats were photographed on days 0 and 14 to record the wound healing status.

[0161] like Figure 9 As shown, after 14 days of treatment with HYP group hydrogel, the wound on the back of the rat was almost completely healed, while obvious wounds were still visible in the blank group and the HY blank hydrogel control group without spirulina bioactive peptide. This indicates that hydrogel containing spirulina bioactive peptide can significantly promote the healing of rat skin wounds.

Claims

1. The application of a multi-component hydrogel containing spirulina bioactive peptides in the preparation of antibacterial and wound-healing active drugs, characterized in that: The solution comprises a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides. Both the oxidized carboxymethyl cellulose and carboxymethyl chitosan aqueous solutions are prepared using PBS buffer solution at pH 7.

4. The volume ratio of the oxidized carboxymethyl cellulose and carboxymethyl chitosan aqueous solutions is 3-7:3-7; the volume ratio of the sum of the volumes of the oxidized carboxymethyl cellulose and carboxymethyl chitosan aqueous solutions to the spirulina bioactive peptides aqueous solution is 10-15:

1.

2. The application of the multi-component hydrogel containing spirulina bioactive peptides according to claim 1 in the preparation of antibacterial and wound-healing active drugs, characterized in that: It also includes sodium carboxymethyl β-glucan; the mass ratio of sodium carboxymethyl β-glucan to the combined mass of aqueous solutions of carboxymethyl cellulose, carboxymethyl chitosan, and spirulina bioactive peptides is 5~8:

100.

3. The application of the multi-component hydrogel containing spirulina bioactive peptides according to claim 1 in the preparation of antibacterial and wound-healing active drugs, characterized in that: The oxidized carboxymethyl cellulose was prepared according to the following steps: Sodium periodate was dissolved in water, and the pH was adjusted to 4.8-5.

2. Carboxymethyl cellulose was added and mixed evenly. The mixture was stirred at 30-35°C in the dark for 4-6 hours. Ethanol was added and stirred for 1-2 hours to obtain a reaction solution. The mixture was poured into the first methanol and stirred for 10-30 minutes. The mixture was filtered, and the solid was washed with the second methanol and dried to obtain oxidized carboxymethyl cellulose. The mass ratio of sodium periodate, water, carboxymethyl cellulose, ethanol, first methanol, and second methanol is 6~7:150:5~5.5:100~120:450~550:50~80.

4. The application of the multi-component hydrogel containing spirulina bioactive peptides according to claim 1 in the preparation of antibacterial and wound-healing active drugs, characterized in that: The oxidation degree of the oxidized carboxymethyl cellulose is 35%~40%.

5. The application of a multi-component hydrogel containing spirulina bioactive peptides according to claim 1 in the preparation of an antibacterial and wound-healing active drug, characterized in that: The degree of carboxylation of the carboxymethyl chitosan is ≥80%.

6. The application of a multi-component hydrogel containing spirulina bioactive peptides according to claim 2 in the preparation of an antibacterial and wound-healing active drug, characterized in that: The degree of substitution of carboxymethyl β-glucan sodium is 60-70%.

7. The application of a multi-component hydrogel containing spirulina bioactive peptides according to claim 1 in the preparation of an antibacterial and wound-healing active drug, characterized in that: The multi-component hydrogel was prepared according to the following steps: Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use; Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution at pH 7.4, and set aside for later use; Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides for later use; A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides were mixed in a volume ratio and stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptides.

8. The application of a multi-component hydrogel containing spirulina bioactive peptides according to claim 2 in the preparation of an antibacterial and wound-healing active drug, characterized in that: The multi-component hydrogel was prepared according to the following steps: Prepare a 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose using PBS buffer solution with pH=7.4, and set aside for later use; Prepare a 5% (w / w) aqueous solution of carboxymethyl chitosan using PBS buffer solution at pH 7.4, and set aside for later use; Prepare a 0.1% (w / w) aqueous solution of spirulina bioactive peptides for later use; A 10% (w / w) aqueous solution of oxidized carboxymethyl cellulose, a 5% (w / w) aqueous solution of carboxymethyl chitosan, and a 0.1% (w / w) aqueous solution of spirulina bioactive peptides were mixed in a volume ratio to obtain a mixed solution, which was then weighed and set aside for later use. Sodium carboxymethyl β-glucan was added to the mixture and stirred until homogeneous to obtain a multi-component hydrogel containing spirulina bioactive peptides; The mass ratio of sodium carboxymethyl β-glucan to the mass of the mixed solution is 5~8:100.

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