Preparation and application of a new type of bacteriostatic and hemostatic composite wound dressing

Abstract

The present application relates to the technical field of biological new material, and more particularly to a preparation and application of a new type of bacteriostatic and hemostatic composite wound dressing, wherein the wood horse-shaped bacillus has a Latin name of Komagataeibacter hansenii and a preservation number of CCTCC NO: M2022186. The present application also relates to the preparation of the wound dressing and the effect of promoting coagulation and inhibiting bacteria when the wound dressing is used. The wound dressing is applied to the medical field of wound hemostasis, bacteriostasis and healing in vitro.

Description

Technical Field

[0001] This invention relates to the field of new biomaterials technology, and in particular to the preparation and application of a novel antibacterial and hemostatic composite wound dressing. Background Technology

[0002] Traditional dressings used in clinical practice mainly include medical absorbent cotton gauze, cotton pads, and petroleum jelly gauze, which provide simple protection and prevent infection. However, traditional dressings cannot keep the wound moist, leading to slow wound healing; the dressing fibers cause a foreign body sensation when used, easily combine with newly formed granulation tissue on the wound surface, resulting in scarring, and are difficult to remove, causing pain to the patient.

[0003] Bacterial cellulose (BC) is a high-molecular-weight biological extracellular polysaccharide synthesized by certain bacteria, composed of glucose monomer molecules polymerized by β-1,4-glycosidic bonds. In addition to its excellent biocompatibility and biodegradability, it also possesses unique properties such as a nano-network structure, high purity, high tensile strength, and high water-holding capacity.

[0004] Coptis chinensis ( C. chinensis It is a common antibacterial and anti-inflammatory traditional Chinese medicine, and has long been used clinically to treat intestinal bacterial infections, reduce fever and relieve pain. It is also one of the most common Chinese medicine materials. Modern medicine has proven that its special pharmacological properties are mainly related to the alkaloids contained in its rhizome, including berberine, coptisine, and other alkaloids.

[0005] This invention aims to solve the problems of traditional wound dressings, such as poor biocompatibility leading to easy wound adhesion, slow healing, and difficulty in wound clotting. Summary of the Invention

[0006] The purpose of this invention is to provide a novel antibacterial and hemostatic composite wound dressing for use in the medical field of wound hemostasis, antibacterial action, and healing of external trauma.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a strain of *Hanthropoietinus*, with the Latin name... Komagataeibacter hansenii The accession number is CCTCC NO: M2022186.

[0009] This invention also provides a process for the production of bacterial cellulose by *Hancrospira haneri*.

[0010] The present invention further provides a novel antibacterial and hemostatic composite wound dressing, comprising the bacterial cellulose and Coptis chinensis.

[0011] Preferably, the bacterial cellulose is a fermentation product of Bacillus hannivarus.

[0012] Preferably, the Coptis chinensis is an aqueous extract of Coptis chinensis.

[0013] Preferably, the ratio of the Coptis chinensis water extract is 15 to 25:1.

[0014] Preferably, the mass fraction of the Coptis chinensis water extract is 10-70%.

[0015] Preferably, the preparation method of the novel antibacterial and hemostatic composite wound dressing is characterized by comprising the following steps:

[0016] (1) Inoculate Hansori bacillus into the culture medium, culture, sterilize, and obtain bacterial cellulose;

[0017] (2) Mix the bacterial cellulose obtained in step (1) with the Coptis chinensis water extract.

[0018] Preferably, the HS culture medium comprises: 1-3% glucose, 0.2-0.8% yeast extract, 0.2-0.8% tryptone, 0.24-0.31% disodium hydrogen phosphate and 0.112-0.118% citric acid, with the balance being water.

[0019] Preferably, the culture temperature is 25–35°C, the culture pH is 6–7, and the culture time is 4–11 days.

[0020] Preferably, the sterilization is performed by mixing the Hansori bacillus ferment with NaOH solution and then autoclaving, wherein the autoclaving temperature is 118–124°C and the autoclaving time is 15–25 min.

[0021] Compared with the prior art, the present invention has the following technical effects:

[0022] The procoagulant and hemostatic properties embodied in this invention are derived from Coptis chinensis extract in the composite material. However, no studies have yet proven that Coptis chinensis extract has procoagulant and hemostatic effects. In this study, the bacterial cellulose-Coptis chinensis extract composite material exhibits excellent procoagulant properties. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The morphology of BC-50% Coptis chinensis extract composite material (BC-CC5) is shown in the figures: a: wet film; b: dry film.

[0025] Figure 2 SEM images of BC-Coptis extract composites: a: BC; b: Coptis extract; cf: BC loaded with 10%, 30%, 50% and 70% Coptis extract.

[0026] Figure 3 FT-IR of BC-Coptis extract composite material;

[0027] Figure 4 The antibacterial properties of BC-Coptis chinensis extract composite material against three bacteria: a) Staphylococcus aureus; b) Pseudomonas aeruginosa; c) Escherichia coli; Standard drug: silver sulfadiazine cream.

[0028] Figure 5 The procoagulant (total coagulation) index of the BC-Coptis extract composite material.

[0029] Biological Preservation Instructions

[0030] Hansori bacillus, Latin name Komagataeibacter hansenii ;

[0031] This strain is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China; deposited on March 3, 2022; accession number: CCTCC NO: M2022186. Detailed Implementation

[0032] This invention provides a bacterium called *Hanthoriformis*, with the Latin name... Komagataeibacter hansenii The accession number is CCTCC NO: M2022186.

[0033] This invention also provides the application of *Hancrospirobacter hansori* in producing bacterial cellulose.

[0034] The present invention further provides a novel antibacterial and hemostatic composite wound dressing, comprising the bacterial cellulose and Coptis chinensis.

[0035] In this invention, the bacterial cellulose is a fermentation product of Bacillus hannivarum.

[0036] In this invention, the Coptis chinensis is preferably an aqueous extract of Coptis chinensis.

[0037] In this invention, the ratio of the Coptis chinensis water extract is 15-25:1; preferably 17-23:1; more preferably 19-21:1; and even more preferably 20:1.

[0038] In this invention, the mass fraction of the Coptis chinensis water extract is 10-70%; preferably 30-70%; more preferably 30-60%; and even more preferably 50%.

[0039] In this invention, the preparation method of a novel antibacterial and hemostatic composite wound dressing is as follows: inoculate Hansori bacillus into a culture medium, culture, sterilize, and obtain bacterial cellulose; mix the obtained bacterial cellulose with Coptis chinensis water extract.

[0040] In this invention, the HS culture medium comprises: 1-3% glucose, 0.2-0.8% yeast extract, 0.2-0.8% tryptone, 0.24-0.31% disodium hydrogen phosphate, and 0.112-0.118% citric acid, with the balance being water; preferably, it comprises 1.3-2.7% glucose, 0.3-0.7% yeast extract, 0.3-0.7% tryptone, 0.26-0.29% disodium hydrogen phosphate, and 0.113-0.31% citric acid, with the balance being water. 0.117% citric acid, balance being water; more preferably 1.7–2.3% glucose, 0.4–0.6% yeast extract, 0.4–0.6% tryptone, 0.26–0.27% disodium hydrogen phosphate and 0.114–0.116% citric acid, balance being water; more preferably 2% glucose, 0.5% yeast extract, 0.5% tryptone, 0.28% disodium hydrogen phosphate and 0.115% citric acid, balance being water.

[0041] In this invention, the culture temperature is 25–35°C; preferably 27–33°C; more preferably 29–31°C; and even more preferably 30°C.

[0042] In this invention, the pH of the culture is 6-7; preferably 6.1-6.9; more preferably 6.3-6.7; and even more preferably 6.5.

[0043] In this invention, the culture time is 4 to 11 days; preferably 5 to 10 days; more preferably 7 to 9 days; and even more preferably 8 days.

[0044] In this invention, the sterilization is performed by mixing the Hansori bacillus ferment with NaOH solution and then autoclaving.

[0045] In this invention, the temperature for autoclaving is 118–124°C; preferably 119–123°C; more preferably 120–122°C; and even more preferably 121°C.

[0046] In this invention, the autoclaving time is 15-25 min; preferably 17-23 min; more preferably 19-21 min; and even more preferably 20 min.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Preparation of BC-Coptis extract composite material

[0050] Hansori Komagataeibacter hansenii Single colonies were picked up using an inoculation loop and transferred to HS medium, the main components of which included 2% (w / v) glucose, 0.5% (w / v) yeast extract, 0.5% (w / v) tryptone, 0.27% (w / v) disodium hydrogen phosphate, and 0.115% (w / v) citric acid. The culture temperature was 30℃, pH 6.5, and the culture was static for 7 days. The white film (BC) at the gas-liquid junction of the bacterial culture was removed using tweezers and autoclaved in a 1% (w / v) NaOH aqueous solution (121℃, 20 min) for later use.

[0051] Preparation method of the compound bacterial cellulose-Coptis extract material in Example 1:

[0052] BC films were added to 10%, 30%, 50%, and 70% aqueous solutions of Coptis chinensis extract, respectively, and shaken at 37℃ and 180 rpm for 24 h to obtain bacterial cellulose-Coptis chinensis extract composites BC-CC1, BC-CC3, BC-CC5, and BC-CC7. The morphology of BC-CC5 wet and dry films is shown below. Figure 1 As shown.

[0053] Example 2

[0054] Characterization of BC-Coptis extract composite material

[0055] SEM characterization of composite materials:

[0056] The freeze-dried BC-Coptis extract obtained in Example 1 was immobilized onto a conductive adhesive substrate on a target stage. After vacuum sputtering for 1 minute in a gold sprayer, it was used for SEM analysis. The microstructure and fiber structure of the composite material were observed at 10 kV. The results are as follows: Figure 2 2a is BC, exhibiting a regular reticular fibrous structure; 2b is Coptis chinensis extract, appearing as irregular lumps. Figure 2 c, d, e, and f are SEM images of the BC-CC1, BC-CC3, BC-CC5, and BC-CC7 composite materials, respectively. The Coptis chinensis extract is uniformly distributed in the pores of the bacterial cellulose network structure, proving that the Coptis chinensis extract was successfully loaded into the bacterial cellulose.

[0057] Example 3

[0058] FT-IR characterization of composite materials:

[0059] The bacterial cellulose and BC-Coptis extract composite material obtained in Example 1 was freeze-dried to constant weight, then placed in a mortar and ground together with dried potassium bromide at a ratio of 1:50. The mixture was then compressed using a tablet press for 2 minutes. The chemical structure of the tablets was analyzed by FT-IR. Test conditions: Scan range 4500–400 cm⁻¹ -1 The resolution is 2 or 4 cm. -1 The number of scans was 32 or 64. The results are as follows: Figure 3 As shown, the BC-Coptis chinensis extract composite material retains almost all the characteristic peaks of bacterial cellulose: 3348, 2897, 1639, 1162, 1060 cm⁻¹. -1 Wait, 3347cm -1 The strong absorption peak at 2894 cm⁻¹ represents the stretching vibration peak of OH, which is caused by hydrogen bonds between cellulose molecules; -1 The absorption peak at 1626 cm⁻¹ is caused by the stretching vibration peak of the CH group; -1 The peak at 1164 cm⁻¹ represents the characteristic absorption peak of the glucose carbonyl (CO) group in cellulose, while the peak at 1164 cm⁻¹ represents the characteristic absorption peak of the CO group in cellulose. -1 This indicates the vibrational peak of the COC functional group; while at 1800~1000 cm⁻¹... -1 Within the specified range, the BC-Coptis extract composite spectrum retains both the characteristic peaks of bacterial cellulose and almost all the characteristic peaks of berberine hydrochloride (berberine), the main component of Coptis chinensis, including: 1603, 1567, 1508, 1482, 1387, 1233, and 1103 cm⁻¹. -1 This further confirms that Coptis chinensis extract has been successfully included in BC.

[0060] Example 4

[0061] Application of BC-Coptis extract composite material

[0062] Antibacterial properties of BC-Coptis extract composite material:

[0063] The antibacterial activity of the BC-Coptis chinensis extract composite material obtained in Example 1 was tested using the agar diffusion method: Disc-shaped bacterial cellulose-Coptis chinensis extract composite materials of various concentrations with a diameter of 6 mm were prepared according to this embodiment. Silver sulfadiazine cream (a commercially available antibacterial burn dressing) was used as a positive control, and BC membrane was used as a negative control. The disc-shaped BC-Coptis chinensis extract composite materials, negative and positive control discs were affixed to LB solid medium containing Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, and cultured overnight. The diameter of the inhibition zone around the disc was observed and recorded. The results are as follows: Figure 4As shown in Table 1, BC had no inhibitory effect on Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The BC-Coptis chinensis extract composite materials at all concentrations also showed no inhibitory effect on Pseudomonas aeruginosa, but exhibited a good inhibitory effect on Staphylococcus aureus. BC-CC3 showed the strongest inhibitory effect on Staphylococcus aureus, reaching 12.38±1.3 mm. The inhibitory effect of the BC-Coptis chinensis extract composite materials at all concentrations on Escherichia coli was not significant; the highest concentration, BC-CC7, showed an inhibition zone diameter of only 8.7±0.7 mm against Escherichia coli. This indicates that the bacterial cellulose-Coptis chinensis extract composite material has a better inhibitory effect on Gram-positive bacteria.

[0064] Table 1. Statistical analysis of the antibacterial zones of the BC-Coptis chinensis extract composite material.

[0065]

[0066] Example 5

[0067] Test of the procoagulant properties of BC-Coptis chinensis extract composite material (whole blood coagulation test):

[0068] BC samples measuring 2cm×2cm, composite materials of BC-Coptis chinensis extract at various concentrations, and gelatin sponges with hemostatic effects (positive control drugs) were placed in disposable culture dishes and heated to 37℃ for 10 minutes.

[0069] Add 100 µL of SD rat blood containing the anticoagulant sodium citrate (volume ratio 9:1) and 10 µL of 0.2 M CaCl2 to the center of each culture dish, and incubate at 37 °C for 10 min. Then, add 25 µL of distilled water to each dish and shake at 37 °C and 30 rpm for 10 min. During shaking, any red blood cells that did not clot and did not adhere to the material were lysed with distilled water.

[0070] 100 µL of the hemoglobin solution from the above steps was pipetted into a 48-well plate. The absorbance (OD) value of the hemoglobin solution was measured at 541 nm using a microplate reader and converted to an Abs value. A lower BCI value indicates better hemostatic performance of the dressing. Results are as follows... Figure 5 As shown, the BCI value of BC was approximately 77%, while the BCI values ​​of the BC-Coptis extract composites at various concentrations, from low to high, were approximately 52%, 47%, 64%, and 95%, respectively, while the BCI value of the gelatin sponge was approximately 63%. This indicates that low concentrations of BC-Coptis extract have better procoagulant properties than the positive control group, and BC-30% Coptis extract showed the best procoagulant effect.

[0071] Example 6

[0072] The BC-Coptis chinensis extract composite material obtained in Example 1 was tested for its procoagulant properties (in vitro coagulation experiment).

[0073] 50 mg of BC, gelatin sponge (positive control group), and 50 mg of BC-Coptis chinensis extract composites of various concentrations were weighed and placed in 24-well plates. After heating in a 37°C water bath for 5 min, 0.50 mL of anticoagulated rabbit blood and 0.1 mL of 0.2 mol / L calcium chloride solution were added to each well of the 24-well plate sequentially. The wells were tilted every 15 s to observe the degree of blood coagulation until the blood stopped flowing, and the coagulation time was recorded. The blank control group received no hemostatic material, only calcium chloride solution, while the positive control group received the same volume of gelatin sponge and calcium chloride solution. Each group of materials was tested in triplicate, and the average value was taken. The results showed that the coagulation effect of BC-Coptis chinensis extract composites of various concentrations was better than that of gelatin sponge (Table 2), with BC-CC3 showing the shortest in vitro coagulation time, requiring only 3.5 ± 0.38 min. BC did not show any coagulation effect, consistent with previous whole blood coagulation experiments.

[0074] Table 2. Statistics on coagulation and hemostasis time of BC-Coptis extract composite material

[0075]

[0076] Example 7

[0077] The BC-Coptis chinensis extract composite material obtained in Example 1 was tested for its procoagulant properties (in vivo tail amputation hemostasis experiment in rats).

[0078] The tails of anesthetized SD rats were severed 5 cm from the tip using a scalpel. The first drop of blood was absorbed with filter paper. A 1 cm × 1 cm sample of BC-Coptis chinensis extract composite material of various concentrations, gelatin sponge (positive control group), and BC were applied to the wound. A blank control group was also included. The rat tail blood drop test was performed using filter paper. The filter paper was changed every 30 seconds after the start of the test, and the bleeding was observed. Hemostasis was considered complete when no more blood dripped onto the filter paper, and the bleeding time was recorded. The results are shown in Table 2. The hemostatic effect of BC-Coptis chinensis extract at all concentrations was superior to that of gelatin sponge, with BC-CC3 showing the fastest hemostatic speed, requiring only 2.5 ± 0.23 min. Bacterial cellulose did not have a hemostatic effect. This also confirms the results of the whole blood coagulation test and the in vitro coagulation test.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bacteriostatic, hemostatic composite wound dressing, characterized in that, Bacterial cellulose fermented by Hippobacterium hippodaleum Komagataeibacter hansenii and water extract of Coptis chinensis with mass fraction of 10%-30% are prepared; the preservation number of the Hippobacterium hippodaleum is CCTCC NO: M2022186; the specification of the water extract of Coptis chinensis is 15-25:

1.

2. The method of producing a composite wound dressing according to claim 1, characterized in that The method comprises the following steps: (1) inoculating Komagataeibacter hansenii into a culture medium, culturing, sterilizing, and obtaining bacterial cellulose; (2) mixing the bacterial cellulose obtained in step (1) with a water extract of Coptis chinensis with a mass fraction of 10%-30%.

3. The production method according to claim 2, characterized by, The culture medium is HS culture medium, which comprises 1%-3% glucose, 0.2%-0.8% yeast extract, 0.2%-0.8% tryptone, 0.24%-0.31% disodium hydrogen phosphate and 0.112%-0.118% citric acid, and the rest is water.

4. The preparation method according to claim 2, characterized in that, The temperature of the culture is 25-35 DEG C, the pH of the culture is 6-7, and the culture time is 4-11 days.

5. The preparation method according to claim 2, characterized in that, The sterilization is high-pressure sterilization after mixing the Komagataeibacter hansenii fermentation product with a NaOH solution, the temperature of the high-pressure sterilization is 118-124 DEG C, and the time of the high-pressure sterilization is 15-25 min.

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