Chitosan sponge material containing allicin as well as preparation method and application of chitosan sponge material

Abstract

The invention discloses a chitosan sponge material containing allicin as well as a preparation method and application of the chitosan sponge material, and belongs to the technical field of preparation of medical materials. The method comprises the following steps: dissolving chitosan in an acetic acid solution, and stirring until the chitosan is completely dissolved to obtain a chitosan solution; adding pentaerythritol glycidyl ether into the chitosan solution, and fully stirring and uniformly mixing to obtain a mixed solution; and adding the allicin into the mixed solution, fully stirring and uniformly mixing, pouring into a mold, and freeze-drying to obtain the chitosan sponge material containing the allicin. The invention provides a chitosan sponge material containing allicin as well as a preparation method and application of the chitosan sponge material. The sponge material has good water absorption and rapid blood coagulation capability.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical material preparation, and in particular relates to a chitosan sponge material containing allicin and a preparation method and application thereof. Background Art

[0002] Uncontrolled massive bleeding is the main cause of death and severe trauma. Statistics show that about one-third of trauma is caused by massive bleeding. In addition, long-term exposure of wounds to the air not only increases the risk of infection, but may also cause serious local wound infections and systemic diseases. Therefore, it is particularly urgent to develop an efficient and biocompatible antibacterial dressing. In surgery and trauma treatment, uncontrolled bleeding remains an important factor affecting patient survival and prognosis. Although commonly used hemostasis methods in operating rooms include ligation of blood vessels, electrocautery, and gauze compression hemostasis, these methods may cause great damage to surrounding tissues while achieving hemostasis. With the widespread use of antimicrobial drugs, pathogens have evolved a high level of multidrug resistance, resulting in the persistence and spread of difficult-to-treat infections, which poses challenges to traditional antimicrobial treatments.

[0003] Allicin, a natural defensive sulfide found in garlic and other Allium plants, has shown antibacterial and antifungal activity and has shown potential in the treatment of diabetes, cardiovascular disease and cancer. Chitosan, a natural biopolymer extracted from the shells of crustaceans, is biocompatible and can promote tissue growth and accelerate wound healing. Its efficient gel-forming properties and the ability to be made into sponge dressings give it a wide range of application prospects in the medical field. At present, several patents have been published for double-layer coagulation dressings prepared by mixing chitosan and other materials as raw materials. These dressings can form a physical blocking barrier with mechanical support when wet and promote blood clot formation. However, these dressings have poor stability in a wet state, and chitosan dressings alone have limited antibacterial effects. Summary of the invention

[0004] The invention aims to provide a chitosan sponge material containing allicin and a preparation method and application thereof. The sponge material has good water absorption and rapid blood coagulation ability.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a chitosan sponge material containing allicin comprises the following steps:

[0007] S1. preparing a chitosan solution: dissolving chitosan in an acetic acid solution, and stirring until the chitosan is completely dissolved to obtain a chitosan solution;

[0008] S2, adding pentaerythritol glycidyl ether to the chitosan solution obtained in step S1, stirring and mixing well to obtain a mixed solution;

[0009] S3, adding allicin to the mixed solution obtained in step S2, fully stirring and mixing, pouring into a mold, and freeze-drying to obtain a chitosan sponge material containing allicin.

[0010] Preferably, in step S1, the volume fraction of the acetic acid solution is 1%-2%.

[0011] Preferably, in step S1, the chitosan concentration is 1%-2%.

[0012] Preferably, in step S2, the molar ratio of pentaerythritol glycidyl ether to monosaccharide molecules in chitosan is 0.002:1 to 0.02:1.

[0013] Preferably, in step S2, the stirring time is 3h-5h.

[0014] Preferably, in step S3, the concentration of allicin is 6 mM.

[0015] Preferably, in step S3, the specific parameters of freeze drying are: freezing temperature is -57°C, and freezing time is 24h.

[0016] Preferably, in step S3, the material of the mold is polypropylene.

[0017] The invention also provides a chitosan sponge material containing allicin prepared by the preparation method.

[0018] The present invention also provides a chitosan sponge material containing allicin prepared by the preparation method or use of the chitosan sponge material containing allicin in preparing a coagulation and antibacterial dressing.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] The invention discloses a chitosan sponge material containing allicin, a preparation method and an application thereof. The method prepares a biocompatible material with excellent antibacterial performance by cross-linking pentaerythritol glycidyl ether with chitosan under acidic conditions. The safe cross-linking agent and the natural organic sulfur compound allicin used in the method give the material an efficient antibacterial effect on Staphylococcus aureus and Escherichia coli, while maintaining the characteristics of low cost and low toxicity. The preparation process is simple and easy to operate, and the prepared sponge material has good water absorption and rapid blood coagulation ability.

[0021] In addition, the material uses a polypropylene mold to prepare sponge materials of different shapes to meet the needs of different wounds. In summary, the sponge material of the present invention has a wide range of application prospects in the preparation of coagulation and antibacterial dressings, especially in emergency medical situations where rapid coagulation and infection prevention are required, showing its unique advantages and potential.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The sponge material provided in Example 1 and Comparative Example 1 is in the form after soaking, wherein: Figure 1 A in the figure is the state of soaking for 0 hours. Figure 1 B in the figure is the state after soaking for 0.3h. Figure 1 C in the figure is the state after soaking for 1h. Figure 1 D in the figure is the state after immersion for 2 hours. Figure 1 E in the figure is the form after immersion for 3 hours;

[0024] Figure 2 A statistical graph of the water absorption rate of the chitosan sponge material containing allicin provided in Examples 1-4;

[0025] Figure 3 A statistical graph of the water absorption rate of the chitosan sponge material containing allicin provided in Examples 5-8;

[0026] Figure 4 A statistical graph of the blood absorption time of the chitosan sponge material containing allicin provided in Examples 1-4;

[0027] Figure 5 A statistical graph of the blood absorption time of the chitosan sponge material containing allicin provided in Examples 5-8;

[0028] Figure 6 A schematic diagram of blood coagulation;

[0029] Figure 7 It is a statistical chart of coagulation index;

[0030] Figure 8 The experimental results of the sponge material provided in Example 1 on the inhibition zone of E. coli;

[0031] Fig. 9 The results of the antibacterial zone test of the sponge material provided in Example 1 on S. aureus;

[0032] Fig.10 The results of the experiment on the E. coli bacteria coated plate using the sponge material provided in Example 1;

[0033] Fig.11The results of the experiment on the sponge material provided in Example 1 against S. aureus bacteria on a plate;

[0034] Fig.12 A statistical graph showing the survival rate of E. coli bacteria on the sponge material provided in Example 1;

[0035] Fig.13 This is a statistical graph of the survival rate of S. aureus bacteria by the sponge material provided in Example 1. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0037] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0038] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0039] Embodiment 1 This embodiment provides a chitosan sponge material containing allicin, and the preparation method comprises the following steps:

[0040] S1. Preparation of chitosan solution: 0.3 g chitosan (viscosity 100 mPa.s, deacetylation degree ≧ 95%) was dissolved in 30 mL of 2% acetic acid solution with a mechanical stirrer turned on in advance, and stirred until the chitosan was completely dissolved to obtain a chitosan solution with a concentration of 1%;

[0041] S2, weighing 1.3 mg, 0.002 eq of pentaerythritol glycidyl ether, adding it to the chitosan solution obtained in step S1, stirring thoroughly for 3 h to mix evenly, to obtain a mixed solution;

[0042] S3. Add 6 mM allicin to the mixed solution obtained in step S2, stir and mix thoroughly, pour into a polypropylene mold, and freeze-dry at -57°C for 24 hours to obtain a chitosan sponge material containing allicin.

[0043] The preparation method of Example 2 is the same as that of Example 1, except that the amount of pentaerythritol glycidyl ether added is 3.3 mg (0.005 eq).

[0044] The preparation method of Example 3 is the same as that of Example 1, except that the amount of pentaerythritol glycidyl ether added is 6.7 mg (0.01 eq).

[0045] The preparation method of Example 4 is the same as that of Example 1, except that the amount of pentaerythritol glycidyl ether added is 13.4 mg (0.02 eq).

[0046] Embodiment 5 This embodiment provides a chitosan sponge material containing allicin, and the preparation method comprises the following steps:

[0047] S1. Preparation of chitosan solution: 0.2 g chitosan (viscosity 100 mPa.s, deacetylation degree ≧ 95%) was dissolved in 10 mL of 2% acetic acid solution with a mechanical stirrer turned on in advance, and stirred until the chitosan was completely dissolved to obtain a chitosan solution with a concentration of 2%;

[0048] S2, weighing 0.9 mg, 0.002 eq of pentaerythritol glycidyl ether, adding them to the chitosan solution obtained in step S1, stirring them thoroughly for 3 h to obtain a mixed solution;

[0049] S3. Add 6 mM allicin to the mixed solution obtained in step S2, stir and mix thoroughly, pour into a polypropylene mold, and freeze-dry at -57°C for 24 hours to obtain a chitosan sponge material containing allicin.

[0050] The preparation method of Example 6 is the same as that of Example 5, except that the amount of pentaerythritol glycidyl ether added is 2.2 mg (0.005 eq).

[0051] The preparation method of Example 7 is the same as that of Example 5, except that the amount of pentaerythritol glycidyl ether added is 4.4 mg (0.01 eq).

[0052] The preparation method of Example 8 is the same as that of Example 5, except that the amount of pentaerythritol glycidyl ether added is 8.9 mg (0.02 eq).

[0053] The preparation method of Comparative Example 1 is the same as that of Example 1, except that pentaerythritol glycidyl ether is not added.

[0054] The effect of the sponge material provided in the above embodiment was verified by the following test:

[0055] 1. Solubility test

[0056] The sponge materials (length, width and height of 1 cm×1 cm×0.5 cm) provided in Example 1 and Comparative Example 1 were placed in glass culture dishes, respectively, and then 50 ml of deionized water was added. The results are as follows: Figure 1 .

[0057] Depend on Figure 1It can be seen that the sponge product without PEGE in Comparative Example 1 dissolved, while the sponge containing PEGE in Example 1 remained a whole and gel-like after soaking for 3 hours, that is, Example 1 became a swollen sponge.

[0058] 2. Liquid absorption test

[0059] (1) Take the blood coagulation sponge products prepared in Examples 1-8 respectively, ensure that they are in a dry state and weigh them, record them as W dry (g).

[0060] (2) Deionized water is added to the sponge product until the sponge is unable to absorb excess water and no excess water overflows from the sponge product.

[0061] (3) Use a straw to absorb the water that cannot be absorbed and weigh it, record it as W wet (g).

[0062] (4) The water absorption rate of each product was calculated using the following formula. The results are shown in Table 1 and Figure 2-Figure 3 ;

[0063]

[0064] Table 1. Water absorption of covalently cross-linked chitosan sponges

[0065]

[0066]

[0067] 3. Blood absorption time

[0068] Take a 1 cm × 1 cm piece of sponge product of Examples 1-8, add 100 uL of fresh chicken blood to the sponge products of Examples 1-8 respectively, and use a timer to time the time for the sponge to absorb blood. The results are shown in Table 2 and Figure 4-Figure 5 .

[0069] Table 2. Blood absorption time of covalently cross-linked chitosan sponges

[0070] Grouping Blood absorption time Example 1 5 Example 2 5 Example 3 5.3 Example 4 5.3 Example 5 7.2 Example 6 6.5 Example 7 6.3 Example 8 6.6

[0071] As shown in Table 2, there is no significant difference in the blood absorption time between the sponge materials with chitosan concentration of 1% and the sponge materials with chitosan concentration of 2%.

[0072] 4. Coagulation test

[0073] (1) Example 1 was used to prepare a sponge product with a length, width and height of 1 cm×1 cm×0.5 cm and put it into a glass culture dish, and commercially available coagulation gelatin was used as the experimental control group.

[0074] (2) 0.25 mL of fresh chicken blood (chicken blood: trisodium citrate = 5:1, the concentration of trisodium citrate solution is 3%-5% (w / v)) was added to a blank glass culture dish, a culture dish containing a sponge product, and a culture dish containing commercially available coagulation gelatin, respectively.

[0075] (3) Place each of the above culture dishes in a 37°C incubator and incubate for 5 minutes, then remove the culture dishes.

[0076] (4) At room temperature, slowly add 50 mL of deionized water into the glass dish along the edge of the dish. Try not to affect the coagulation of blood on the sponge and gelatin during the pouring process.

[0077] (5) The unabsorbed red blood cells will be dissolved in the solution, and the absorption value is detected at a wavelength of 540 nm using an ultraviolet spectrophotometer. 0.25 mL of chicken blood in a blank glass culture dish is dissolved in 50 mL of deionized water as a negative control;

[0078] (6) The blood coagulation index (BCI) of the sponge product was calculated using the following formula. The results are shown in Table 3 and Figure 6-Figure 7 .

[0079]

[0080] Table 3. Coagulation index of covalently cross-linked chitosan sponges

[0081] Material Coagulation index (%) Control group 98.73 Example 1 17.18 Commercial blood coagulation gelatin 96.24

[0082] 5. Inhibition zone test

[0083] S. aureus and E. coli were used to evaluate the antibacterial properties of chitosan antibacterial sponge. The volume ratio of bacterial solution to LB liquid culture medium was 1:100 (v / v), and the culture was incubated in a shaker at 37°C and 200 rpm / min for 7 hours. The incubated bacterial solution was washed three times with sterile PBS and then diluted to 10 6 CFU / mL. Place the chitosan sponge under ultraviolet light for 30 minutes for sterilization. Slowly pour 10mL of LB agar medium into a sterile culture dish and wait until it cools and solidifies. Add 100μL of bacterial solution to the LB agar medium and disperse evenly. Use sterile tweezers to place the chitosan sponge on the culture medium with bacterial solution. Place the culture dish in a 37℃ constant temperature incubator and record the results after 24 hours of incubation. The results are as follows Figure 8-9 .

[0084] 6. Bacterial plate count experiment

[0085] The volume ratio of bacterial solution to Luria-Bertani (LB) liquid medium was 1:100 (v / v), and the culture was incubated at 37°C, 200 rpm / min in a shaker for 7 hours. The incubated bacterial solution was washed three times with sterile PBS and diluted again with PBS to 10 6 CFU / mL for later use. Place the chitosan sponge under ultraviolet light for 30 minutes for sterilization, and then place it in a sterile 24-well plate. Add 2mL of sterile PBS to the 24-well plates without chitosan sponge, chitosan sponge, and chitosan sponge / allicin. Add 200μL of bacterial solution to the corresponding wells, and then incubate in a 37℃ constant temperature incubator for 12 hours. After incubation for 12 hours, take out 100μL of bacterial solution from each well and add it to LB agar medium for plating. Place the culture dish in a 37℃ constant temperature incubator and incubate for 24 hours and record the results. The results are as follows. Figure 10-13 .

[0086] From Table 1-3 and Figure 1-Figure 13 It can be seen that the sponge product of the present invention has good water absorption rate and coagulation rate. It has good antibacterial performance against S. aureus and E. coli; and the shorter blood absorption time promotes the aggregation of formed elements in the blood and accelerates the body's own coagulation rate. In addition, it can also achieve physical coagulation with the negative charge carried by red blood cells in the body to form clots; at the same time, the blood clots formed at the wound have the effect of physically blocking the wound, so it can quickly absorb blood and coagulate in time, which has obvious advantages compared with the coagulation gelatin sold on the market.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a chitosan sponge material containing allicin, characterized in that: The steps include: S1. preparing a chitosan solution: dissolving chitosan in an acetic acid solution, and stirring until the chitosan is completely dissolved to obtain a chitosan solution; S2, adding pentaerythritol glycidyl ether to the chitosan solution obtained in step S1, stirring and mixing well to obtain a mixed solution; S3, adding allicin to the mixed solution obtained in step S2, fully stirring and mixing, pouring into a mold, and freeze-drying to obtain a chitosan sponge material containing allicin.

2. The preparation method according to claim 1, characterized in that: In step S1, the volume fraction of the acetic acid solution is 1%-2%.

3. The preparation method according to claim 1, characterized in that: In step S1, the chitosan concentration is 1%-2%.

4. The preparation method according to claim 1, characterized in that: In step S2, the molar ratio of pentaerythritol glycidyl ether to monosaccharide molecules in chitosan is 0.002:1-0.02:

1.

5. The preparation method according to claim 1, characterized in that: In step S2, the stirring time is 3h-5h.

6. The preparation method according to claim 1, characterized in that: In step S3, the concentration of allicin is 6 mM.

7. The preparation method according to claim 1, characterized in that: In step S3, the specific parameters of freeze drying are: freezing temperature is -57°C, and freezing time is 24h.

8. The preparation method according to claim 1, characterized in that: In step S3, the material of the mold is polypropylene.

9. The chitosan sponge material containing allicin prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the chitosan sponge material containing allicin prepared by the preparation method according to any one of claims 1 to 8 or the chitosan sponge material containing allicin according to claim 9 in the preparation of anticoagulant and antibacterial dressings.

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