Natural bio-based hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedical materials, and discloses natural bio-based hydrogel as well as a preparation method and application thereof. Comprising the following preparation steps: performing alkaline modification on konjac glucomannan to obtain deacetylated konjac glucomannan; carrying out ammoniation modification on gelatin to obtain ammoniated gelatin; treating the amomum villosum volatile oil with a cosolvent to obtain an amomum villosum volatile oil dispersion liquid; and mixing the deacetylated konjac glucomannan and ammoniated gelatin, then adding the amomum villosum volatile oil dispersion liquid, and fully reacting to obtain the natural bio-based hydrogel. After the gelatin is ammoniated and modified, the content of free amino groups is increased by about 220%, and the network stability is improved. The energy storage modulus of the prepared hydrogel can reach 38.25 Pa, and the prepared hydrogel is excellent in mechanical property. The scavenging rate of the hydrogel to DPPH free radicals reaches up to 65.9%, and the hydrogel has good oxidation resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medical materials, and more particularly to a natural bio-based hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Bacterial infected wounds are usually accompanied by impaired angiogenesis, overexpression of matrix metalloproteinases (MMPs), and strong inflammatory response of macrophages. These factors lead to abnormal increase of reactive oxygen species (ROS) in vivo, thereby destroying the cell microenvironment, inhibiting the formation of healthy tissues, and seriously hindering wound healing. At present, bacterial infected wounds have become a big challenge in clinical treatment, not only prolonging the treatment period and increasing the probability of complications, but also bringing a huge burden to patients and the medical system. In traditional treatment methods, functional dressings such as functional gauze and tissue glue are widely used, but they have problems such as insufficient flexibility, poor adaptability, and limited antibacterial effect.

[0003] In recent years, hydrogel materials have attracted widespread attention in the field of wound dressings due to their good biocompatibility, high water content, and moldability. Hydrogels can adapt to the shape of the wound, achieve complete coverage of irregular wounds, and thus provide a closed environment to promote wound healing and improve drug release efficiency. However, natural polymer hydrogels such as konjac glucomannan (KGM) have excellent water absorption and swelling properties and certain gel-forming properties, but their mechanical strength is low and their biological activity is limited, making it difficult to meet the treatment needs of complex infected wounds.

[0004] KGM is a natural polymer formed by the connection of D-glucose and D-mannose through β-1, 4-glycosidic bond. The hydrophilicity of its main chain and the presence of acetyl group make it outstanding in thermal reversible gelation. In alkaline conditions, the removal of acetyl group can form a thermal irreversible hydrogel. However, single KGM hydrogel still has deficiencies in dealing with bacterial infection environment.

[0005] Amomum villosum (AV) is a traditional Chinese medicinal material, and its main components include volatile terpenes (such as borneol acetate and camphor), flavonoids, polyphenols, and polysaccharides, which have significant antioxidant, antibacterial, anti-inflammatory, and gastrointestinal function promoting biological activities. However, the effective components of Amomum villosum are complex and mostly volatile or hydrophobic small molecules, which have poor stability, are difficult to disperse uniformly in the hydrophilic system of hydrogel and maintain long-term active release, and are easy to disturb the three-dimensional network structure of hydrogel, thereby affecting the gel performance and mechanical properties. Currently, there is no literature or public technology to disclose the successful realization of efficient compounding of Amomum villosum and KGM and its systematic application in medical dressings. SUMMARY

[0006] The present application provides a natural bio-based hydrogel to overcome the physicochemical incompatibility between the above-mentioned natural sand and KGM, realize the synergistic stable compounding of the two and the like.

[0007] Another object of the present application is to provide a preparation method of the natural bio-based hydrogel.

[0008] Another object of the present application is to provide an application of the natural bio-based hydrogel.

[0009] To solve the above technical problems, the technical scheme of the present application is as follows.

[0010] A preparation method of a natural bio-based hydrogel, comprising the following steps:

[0011] S1, alkali-modifying konjac glucomannan (KGM) to obtain deacetylated konjac glucomannan (dKGM);

[0012] S2, ammoniating gelatin (Gel) to obtain ammoniated gelatin (AGel);

[0013] S3, treating the volatile oil of natural sand (AV) by a cosolvent to obtain a natural sand volatile oil dispersion;

[0014] S4, mixing the deacetylated konjac glucomannan and the ammoniated gelatin, then adding the natural sand volatile oil dispersion, and fully reacting to obtain the natural bio-based hydrogel.

[0015] Preferably, the pH of the konjac glucomannan is 5.0-7.0, the viscosity is ≥15000 mPas, and the purity (calculated on a dry basis) is ≥95%.

[0016] Preferably, the volatile oil of natural sand is treated by Tween 80 cosolvent.

[0017] Further, the concentration of the deacetylated konjac glucomannan is 0.5%-5% w / v, the concentration of the ammoniated gelatin is 1%-10% w / v, and the concentration of the natural sand volatile oil dispersion is 0.5%-5% w / v.

[0018] Preferably, the concentration of the deacetylated konjac glucomannan is 5% w / v.

[0019] Preferably, the concentration of the natural sand volatile oil dispersion is 5% w / v.

[0020] Further, alkali modification is performed with an alkaline inorganic salt, and the alkaline inorganic salt accounts for 10%-25% of the konjac glucomannan.

[0021] Further, in S1, alkali modification is performed with sodium carbonate and sodium bicarbonate, and the mass ratio of sodium carbonate to sodium bicarbonate is 1:2-3.

[0022] Further, in S2, the method for modifying gelatin by amination is that gelatin is reacted with adipic acid dihydrazine in a reaction system to obtain aminated gelatin; the reaction system comprises 1-ethyl-(3-dimethylaminopropyl) carbodiimide, 1-hydroxybenzotriazole and dimethyl sulfoxide mixed solvents.

[0023] Further, the mass ratio of adipic acid dihydrazine to gelatin is 4-5:1.

[0024] Further, the molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide to 1-hydroxybenzotriazole is 1:2-3.

[0025] Further, in the dimethyl sulfoxide mixed solvent, the volume ratio of dimethyl sulfoxide to water is 1:2-3.

[0026] A natural bio-based hydrogel is prepared by the method for preparing the natural bio-based hydrogel.

[0027] The natural bio-based hydrogel is used for preparing a medical dressing.

[0028] The present application constructs a dynamic reversible hydrogel network by compounding deacetylated konjac glucomannan and aminated gelatin, innovatively uses Schiff base reaction to realize multi-component dynamic crosslinking, and improves network crosslinking degree and structural compactness by controlling the deacetylation degree of KGM and the amination efficiency of gelatin. The physical interaction between the hydrophobic volatile components of volatile oil of Amomum villosum and the hydrophilic network constructs a natural bio-based hydrogel with a semi-interpenetrating network structure with enhanced mechanical properties. The main components in Amomum villosum not only provide antioxidant activity, but also serve as physical association points to assist in the micro-stability and elasticity enhancement of the colloidal structure. In addition, unlike conventional Amomum or other aromatic additives, the present application selects Amomum villosum as the source of natural volatile oil, and the unique ingredients such as borneol acetate and camphor in Amomum villosum show outstanding antioxidant performance. The hydrogel network of the present application can stably encapsulate volatile oil of Amomum villosum, and the synergistic fixation between the polymer network and the volatile oil further improves the biological function stability of the hydrogel.

[0029] Compared with the prior art, the beneficial effects of the technical scheme of the present application are:

[0030] The free amino group content of the gelatin modified by amination in the application is increased by about 220%, which significantly increases the number of crosslinking sites and improves the network stability. The storage modulus of the prepared hydrogel can reach 38.25 Pa, and the mechanical property is excellent. The scavenging rate of the hydrogel to DPPH free radicals is as high as 65.9%, which is significantly higher than that of other control groups, and has good antioxidant performance. The hydrogel of the application is shaped completely, does not flow when inverted, and shows good structural stability and self-supporting ability. In addition, the natural network constructed by modified KGM and aminated gelatin can stably encapsulate volatile oil of Amomum villosum, and the system is transparent and uniform, and no precipitation is observed, which shows good dispersion and compatibility characteristics. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 (A) before the formation of the functional hydrogel, (B) after the formation of the functional hydrogel;

[0032] Figure 2 (A) KGM L -AGel-AV L’ , (B) KGM L -AGel-AV H’ , (C) KGM H -AGel-AV L’ , (D) KGM H -AGel-AV H’ . DETAILED DESCRIPTION

[0033] The application will be further described below in conjunction with the drawings and specific examples in the specification, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The konjac glucomannan used in the examples has a pH of 5.0-7.0, a viscosity of ≥15000 mPas, and a purity (on a dry basis) of ≥95%.

[0036] Example 1

[0037] 1 g of Amomum villosum was soaked in 10 mL of anhydrous ethanol, stirred at 500 rpm at room temperature for 24 h. The residue was filtered off and stored at -20°C overnight. The next day, the solvent was removed by rotary evaporation (37°C), and after multiple purifications, Amomum villosum volatile oil was obtained. After the Amomum villosum volatile oil was dissolved in a small amount of Tween 80, deionized water was added to prepare a 0.5% w / v Amomum villosum volatile oil dispersion for use.

[0038] Take konjac glucomannan powder (KGM) 0.50 g, sodium carbonate 0.0125 g, sodium bicarbonate 0.0375 g, add deionized water, magnetic stirring uniform, placed in 95℃ water bath for 1 hour, freeze-dried after dialysis for standby. When using, prepare 0.5% w / v dKGM.

[0039] Take another gelatin 1 g and adipic acid dihydrazine 4 g into 70℃ deionized water, heated and stirred until dissolved. Take 1-hydroxybenzotriazole (HoBt) 1.3 g and EDC·HCl 5.53 g dissolved in DMSO / H2O mixed solvent (volume ratio 1:3), slowly add the gelatin solution, adjust pH to 5.0-5.5, react at room temperature for 24 hours, freeze-dried after dialysis, get aminated gelatin (AGel). When using, prepare 1% w / v AGel.

[0040] Heat the obtained dKGM to 60℃, add the same volume of AGel solution and stir uniformly, then slowly add the sand volatile oil dispersion (0.1 times the total volume) assisted by Tween 80, continue to stir and maintain the temperature at 40-45℃, make it fully dissolved, adjust the pH to 5.0, stand at 4℃ for 12-24 hours, finally get the bio-based hydrogel (dKGM L -AGel-AV L’ )。

[0041] Example 2

[0042] Example 2 technical scheme is similar to example 1, the difference is that the sand volatile oil is dissolved in a small amount of Tween 80, then added to deionized water to prepare a 5% w / v sand volatile oil dispersion for standby. Finally get the bio-based hydrogel (dKGM L -AGel-AV H’ )。

[0043] Example 3

[0044] Example 3 technical scheme is similar to example 1, the difference is that take konjac glucomannan powder (KGM) 0.5 g, sodium carbonate 0.025 g, sodium bicarbonate 0.05 g, add deionized water, magnetic stirring uniform, placed in 95℃ water bath for 1 hour, freeze-dried after dialysis for standby. When using, prepare 5% w / v dKGM. Finally get the bio-based hydrogel (dKGM H -AGel-AV L’ )。

[0045] Example 4

[0046] Example 4 The technical solution is similar to Example 1, except that the volatile oil of Pinellia ternate is dissolved in a small amount of Tween 80, then added to deionized water to prepare a 5% w / v Pinellia ternate volatile oil dispersion for use; 0.5 g of konjac glucomannan powder (KGM), 0.025 g of sodium carbonate, and 0.05 g of sodium bicarbonate are weighed and added to deionized water, then uniformly stirred by magnetic stirring, and placed in a 95°C water bath for 1 hour, then freeze-dried after dialysis for use. When used, a 5% w / v dKGM is prepared. Finally, a bio-based hydrogel (dKGM H -AGel-AV H’ ) is obtained.

[0047] Example 5

[0048] The technical solution of Example 5 is similar to Example 1, except that 0.50 g of konjac glucomannan powder (KGM), 0.0416 g of sodium carbonate, and 0.0834 g of sodium bicarbonate are weighed and added to deionized water, then uniformly stirred by magnetic stirring, and placed in a 95°C water bath for 1 hour, then freeze-dried after dialysis for use. When used, a 0.5% w / v dKGM is prepared.

[0049] Gelatin 1 g and adipic acid dihydrazide 5 g are heated and stirred in deionized water at 70°C until dissolved. 1-Hydroxybenzotriazole (HoBt) 1.3 g and EDC-HCl 3.69 g are dissolved in a DMSO / H2O mixed solvent (volume ratio 1:2), slowly added to the gelatin solution, the pH is adjusted to 5.0-5.5, and the reaction is carried out at room temperature for 24 hours. After dialysis, freeze-drying is performed to obtain aminated gelatin (AGel). When used, a 10% w / v AGel is prepared.

[0050] The obtained dKGM solution is heated to 60°C to fully dissolve, the same volume of AGel solution is added and stirred uniformly, then the Pinellia ternate volatile oil dispersion (0.1 times the total volume) assisted by Tween 80 is slowly added, the stirring is continued and the temperature is maintained at 40-45°C, the pH is adjusted to 5.0, and the final bio-based hydrogel (KGM-AGel H -AV) is obtained by standing at 4°C for 12-24 hours.

[0051] Comparative Example 1

[0052] The technical solution is similar to Example 1, except that the KGM is not alkali-modified.

[0053] Comparative Example 2

[0054] The technical solution is similar to Example 1, except that the Gel is not aminated.

[0055] Comparative Example 3

[0056] The technical solution is similar to Example 1, except that no Pinellia ternate volatile oil is added.

[0057] Comparative Example 4

[0058] The technical solution is similar to that of Example 1, except that 0.5 g of konjac glucomannan powder (KGM), 0.24 g of sodium carbonate, and 0.5 g of sodium bicarbonate are weighed and added to deionized water, and then magnetically stirred until uniform. The mixture is placed in a 95°C water bath for 1 hour, then dialyzed and freeze-dried for later use. When used, it is prepared into a 0.5% w / v dKGM solution.

[0059] Comparative Example 5

[0060] The technical solution is similar to that of Example 1, except that 1 g of gelatin and 8 g of adipic acid dihydrazide are added to deionized water and heated and stirred at 70°C until dissolved. 1.3 g of 1-hydroxybenzotriazole (HoBt) and 5.53 g of EDC-HCl are dissolved in a DMSO / H2O mixed solvent (volume ratio 1:3), and then slowly added to the gelatin solution. The pH is adjusted to 5.0-5.5, and the reaction is carried out at room temperature for 24 hours. After dialysis, the freeze-dried product is obtained as aminated gelatin (AGel).

[0061] Comparative Example 6

[0062] The technical solution is similar to that of Example 1, except that a small amount of Tween 80 is added to assist the dissolution of the volatile oil of Xingren sand, and then added to deionized water to prepare a 0.2% w / v Xingren sand volatile oil dispersion for later use.

[0063] Comparative Example 7

[0064] The technical solution is similar to that of Example 1, except that 1 g of green shell sand is soaked in 10 mL of anhydrous ethanol and stirred at 500 rpm at room temperature for 24 hours. The residue is filtered off and stored at -20°C overnight. The next day, the solvent is removed by rotary evaporation (37°C). After multiple purifications, the green shell sand volatile oil is obtained.

[0065] Detection method

[0066] 1. Determination of mechanical properties

[0067] The sample is placed in the sample loading area of a Malvern Kinexus Pro+ rheometer, and a 20 mm flat plate is selected with a spacing of 1.0 mm. At 25°C, the strain amplitude sweep mode is used to study the rheological properties of the hydrogel. A fixed frequency of 1.0 Hz is used to scan the strain range of 0.1%-800%, which is used to determine the storage modulus and loss modulus of the hydrogel.

[0068] 2. Detection of amino content

[0069] The indanetrione experiment method is combined with ultraviolet spectrophotometry to quantitatively detect the amino content. The specific method is as follows: different concentrations of glycine solution are prepared, and a glycine standard curve is established according to the absorbance at 570 nm. Appropriate amounts of the modified and unmodified gelatin samples are weighed, dissolved, and prepared into aqueous solutions, and the ultraviolet absorbance at 570 nm is recorded. The free amino content in the sample is converted by comparing the standard curve.

[0070]

[0071] X is the free amino content in the unmodified gelatin sample, and Y is the free amino content in the modified gelatin.

[0072] 3. Radical DPPH scavenging rate

[0073] An equal amount of hydrogel (such as 100 μL of liquid or 100 mg of wet glue) is placed in an EP tube; 2 mL of DPPH ethanol solution (0.1 mM) is added; the reaction is carried out in the dark for 30 min (room temperature); the supernatant is aspirated, and the absorbance A at 517 nm is measured s ; the absorbance A0 of the blank control is also measured; and the DPPH scavenging rate is calculated:

[0074] Analysis description

[0075] Figure 1 A is the state of each group of hydrogels before reaction, and the system is a transparent liquid without stratification or turbidity, indicating that the modified raw materials and volatile oil of Ammoniacum have good dispersibility and compatibility in the mixed system. Figure 1 B is the hydrogel formed after the reaction is completed, and all examples are successfully gelled, and the gel is complete, the boundary is clear, has good formability and self-supporting property, and no stratification or separation is observed, indicating that the hydrogel network structure of the application is stable, the gelling efficiency is high, and the components have good synergistic effect.

[0076] Figure 2 The results show that the dKGM L -AGel-AV H ' hydrogel has a higher storage modulus than the dKGM L -AGel-AV L ' hydrogel, and the dKGM H -AGel-AV L ' and the dKGM H -AGel-AV H’ ' hydrogel have a higher storage modulus than the dKGM L -AGel-AV L’ ' and the dKGM L -AGel-AV H’The storage modulus of the hydrogel indicates that increasing the content ratio of dKGM and the content of AV volatile oil can increase the crosslinking density of the hydrogel and enhance the mechanical properties, wherein dKGM H -AGel-AV H’ The storage modulus of the hydrogel can reach 38.25 Pa, and the mechanical properties are excellent.

[0077] Table 1 mechanical properties on the storage modulus

[0078] G' (Pa) Example 1 13.4 Comparative Example 1 8.2 Comparative Example 2 9.4 Comparative Example 3 10.1 Comparative Example 4 10.3 Comparative Example 5 11.6 Comparative Example 6 10.9

[0079] As shown in Table 1, the KGM in Comparative Example 1 is not subjected to alkaline modification, resulting in that the KGM molecular chain retains more acetyl groups and cannot form a stable thermally irreversible gel structure, the elastic response is extremely poor, and the gel is extremely easy to collapse. In Comparative Example 2, the aminated gelatin is not used, the Schiff base reaction is difficult to occur, and the hydrogel network is mainly maintained by physical entanglement or a small amount of hydrogen bond, and G' decreases. In Comparative Example 3, AV volatile oil is not added, although the basic network structure is established, but lacks the assistance of hydrophobic weak association, resulting in loose structure and reduced modulus, indicating that AV volatile oil also plays an important role in the compactness of the microstructure. In Comparative Example 4, the amount of alkaline modification is excessive, resulting in partial degradation of KGM molecules, and excessive deacetylation leads to too high rigidity of the skeleton, and the obtained hydrogel network structure is discontinuous and has insufficient rheological properties. In Comparative Example 5, ADH is used in excess in the system of the present application, resulting in unbalanced, loose and lack of uniformity of the crosslinked network, and finally showing decreased modulus and unstable structure. In Comparative Example 6, the concentration of AV volatile oil is too low to support the formation of effective semi-interpenetrating network, and in the system with low KGM skeleton concentration, the hydrophobic component may disturb the initial formation process of the gel structure due to uneven distribution in the system, thereby causing certain interference to the network stability. Therefore, the concentration of AV volatile oil needs to be synergistically controlled with the concentrations of KGM and AGel to ensure the combination of structural integrity and functionality.

[0080] In addition, under the control of the reaction ratio of ADH and gelatin, the synergistic catalysis of EDC / HoBt, the present application forms a high-quality AGel with uniform structure and stable performance, which provides good support for constructing high-strength natural hydrogel. The amino content is measured to be increased by 220% in Example 5.

[0081] Table 2 DPPH free radical scavenging rate comparison table

[0082] DPPH Scavenging Rate (%) Example 1 52.7 Example 2 60.8 Example 3 58.4 Example 4 65.9 Comparative Example 3 12.5 Comparative Example 6 23.6 Comparative Example 7 34.2 Vitamin C (0.1 mg / mL) 87.5

[0083] The results in Table 2 show that all the examples exhibit good free radical scavenging ability, indicating that they still retain biological functions as antioxidant active components in the hydrogel system. When the concentration of volatile oil of Semen Dalbergiae Hupehensis is higher, the DPPH scavenging rate is as high as 65.9%, while that of Comparative Example 3 is only 17.5% and that of Comparative Example 6 is 23.6%. The hydrogels in the examples not only have antioxidant capacity due to the addition of AV, but more importantly, the dynamic three-dimensional network structure constructed by dKGM and AGel has good drug immobilization, diffusion release and interface stability, significantly improving the loading efficiency of volatile oil and the interface contact efficiency of free radical scavenging, playing a synergistic effect. In addition, the main components of green shell sand used in Comparative Example 7 are eucalyptol, alpha-pinene and other cyclic hydrocarbon and olefin structures, which lack effective antioxidant groups and have weak synergistic ability with the hydrophilic hydrogel network, resulting in low DPPH scavenging rate.

[0084] Obviously, the above examples of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. It is not necessary or possible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A method for preparing a natural bio-based hydrogel, characterized in that: The following steps are involved: S1, alkali-modifying konjac glucomannan to obtain deacetylated konjac glucomannan; S2, ammoniation-modifying the gelatin to obtain ammoniated gelatin; S3, treating the Amomum villosum volatile oil with a cosolvent to obtain an Amomum villosum volatile oil dispersion; S4. Mixing deacetylated konjac glucomannan and ammoniated gelatin, and then adding Amomum villosum volatile oil dispersion, and after sufficient reaction, obtaining a natural bio-based hydrogel.

2. The method for preparing the natural bio-based hydrogel according to claim 1, characterized in that: The concentration of deacetylated konjac glucomannan is 0.5% to 5% w / v, the concentration of ammoniated gelatin is 1% to 10% w / v, and the concentration of the Amomum villosum essential oil dispersion is 0.5% to 5% w / v.

3. The method for preparing the natural bio-based hydrogel according to claim 1, characterized in that: The alkaline modification is carried out with alkaline inorganic salt, and the alkaline inorganic salt accounts for 10% to 25% of the konjac glucomannan.

4. The method for preparing the natural bio-based hydrogel according to claim 2, characterized in that: In S1, alkaline modification is performed with sodium carbonate and sodium bicarbonate, and the mass ratio of sodium carbonate to sodium bicarbonate is 1:2-3.

5. The method for preparing the natural bio-based hydrogel according to claim 1, characterized in that: In S2, the method for ammoniation modification of gelatin is: gelatin and dihydrazide adipic acid react in a reaction system to obtain aminated gelatin; the reaction system includes a mixed solvent of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-hydroxybenzotriazole, and dimethyl sulfoxide.

6. The method for preparing the natural bio-based hydrogel according to claim 5, characterized in that: The mass ratio of dihydrazine adipic acid to gelatin is 4-5:

1.

7. The method for preparing the natural bio-based hydrogel according to claim 5, characterized in that: The molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to 1-hydroxybenzotriazole is 1:2-3.

8. The method for preparing the natural bio-based hydrogel according to claim 5, characterized in that: In the dimethyl sulfoxide mixed solvent, the volume ratio of dimethyl sulfoxide to water is 1:2-3.

9. A natural bio-based hydrogel, characterized in that: The hydrogel is prepared by the method for preparing the natural bio-based hydrogel according to any one of claims 1 to 8.

10. An application of the natural bio-based hydrogel according to claim 9, characterized in that: Used to prepare medical dressings.

Images