Preparation method and application of bletilla striata component extract for improving diabetic foot ulcer pain

Abstract

The invention discloses a preparation method and application of a bletilla striata component extract for improving diabetic foot ulcer pain, and the method comprises the following steps: S1, washing bletilla striata tubers with deionized water, air-drying, and crushing to obtain bletilla striata powder; s2, adding the porous magnetic microsphere immobilized cellulase into deionized water, performing ultrasonic dispersion, adding bletilla striata powder, and performing stirring reaction under ultrasonic assistance and heating; s3, filtering and collecting filtrate 1, adding filter residues into deionized water, stirring and reacting under ultrasonic assistance and heating, filtering and collecting filtrate 2, combining and collecting the filtrate 1 and the filtrate 2, and concentrating to obtain a crude bletilla striata extracting solution; and S4, adding ethanol into the crude bletilla striata extracting solution, stirring, standing overnight, centrifuging, washing precipitate with ethanol, re-dissolving in deionized water, stirring, filtering, ultra-filtering and concentrating the obtained solution, then adding ethanol, stirring, standing overnight, centrifuging, washing precipitate with ethanol, and drying to obtain the bletilla striata component extract.

Description

Technical Field

[0001] The present invention relates to the pharmaceutical field, and particularly to a preparation method and application of a Bletilla striata component extract for improving the pain of diabetic foot ulcers. Background Art

[0002] Bletilla striata is the dried tuber of the orchid plant Bletilla striata, which is bitter, astringent, dry and slightly cold in nature, and has the effects of astringing to arrest bleeding, detumescence and promoting granulation, clearing heat and promoting diuresis, and is widely used in traditional Chinese medicine clinical practice and daily health care. There are various active ingredients in the Bletilla striata tuber, and Bletilla striata polysaccharide is its main extract. The Bletilla striata extract can significantly shorten the bleeding and coagulation time, and its hemostatic effect is related to the colloid contained; the Bletilla striata extract has an obvious protective effect on the gastric mucosa; the Bletilla striata extract can promote granulation growth and wound healing in laboratory burn and scald animal models.

[0003] The traditional extraction processes of the Bletilla striata extract mainly include water extraction method, cellulase hydrolysis method, etc. The water extraction method usually requires high-temperature treatment (the temperature needs to be above 60°C), such as a preparation method of a Bletilla striata extract and its application in preparing antibacterial products disclosed in patent CN116327799A, a preparation method and application of a Bletilla striata extract disclosed in patent CN103536462A, etc. Such processes have the following disadvantages: the starch in Bletilla striata will dissolve into the product during the high-temperature treatment process, resulting in an increase in the impurity content. The cellulase hydrolysis method can achieve extraction at low temperature by adding cellulase to decompose the cell wall, but it will introduce protein impurities into the product, which will also reduce the purity of the product.

[0004] A Bletilla striata polysaccharide extract and its preparation method disclosed in patent CN109970879B can effectively reduce the increase of protein impurities caused by the entry of cellulase into the product by using immobilized cellulase for the extraction of Bletilla striata polysaccharide. It provides a good strategy for application in Bletilla striata extraction, but the preparation method of the immobilized cellulase in this patent is relatively complex, and it is also necessary to provide more alternative solutions.

[0005] Therefore, it is necessary to improve the existing technology now to provide more reliable solutions. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method and application of a Bletilla striata component extract for improving the pain of diabetic foot ulcers in view of the above deficiencies in the existing technology.

[0007] To solve the above technical problem, the technical solution adopted by the present invention is: in the first aspect of the present invention, a preparation method of a Bletilla striata component extract for improving the pain of diabetic foot ulcers is provided, including the following steps:

[0008] S1. Wash the Bletilla striata tuber with deionized water, air-dry it, and then crush it to obtain Bletilla striata powder;

[0009] S2. Add the porous magnetic microsphere-immobilized cellulase to deionized water, ultrasonically disperse it, then add the Bletilla striata powder, and stir and react under ultrasonic assistance and heating;

[0010] S3. Filter and collect filtrate 1. Add the filter residue to deionized water again, stir and react under ultrasonic assistance and heating, filter and collect filtrate 2, combine filtrate 1 and filtrate 2, and concentrate to obtain the crude Bletilla striata extract;

[0011] S4. Add ethanol to the crude Bletilla striata extract, stir, let it stand overnight, centrifuge, wash the precipitate with ethanol, redissolve it in deionized water, stir, first filter the obtained solution with a microfiltration instrument, then perform ultrafiltration, concentrate the obtained ultrafiltrate, then add ethanol, stir, let it stand overnight, centrifuge, wash the precipitate with ethanol, and dry to obtain the Bletilla striata component extract.

[0012] Preferably, the porous magnetic microsphere-immobilized cellulase is prepared by the following method:

[0013] S2-1. Prepare porous magnetic microspheres:

[0014] Take FeSO4·7H2O, FeCl3·6H2O, and polyethylene glycol, add them to deionized water, stir, then adjust the pH value to alkaline with ammonia water, stir and react under heating, then heat and keep warm, cool, wash, and calcine the obtained precursor to obtain porous Fe3O4 magnetic microspheres;

[0015] S2-2. Prepare the immobilized carrier:

[0016] Take the porous Fe3O4 magnetic microspheres, add them to deionized water, and ultrasonically disperse to obtain the carrier dispersion;

[0017] Add citric acid, chitosan, and glucose to the carrier dispersion, stir, transfer the obtained mixture to a reaction kettle, and react under heating. After the reaction, cool, filter, wash, and dry to obtain the immobilized carrier;

[0018] S2-3. Load and immobilize cellulase:

[0019] Take the immobilized carrier, add it to PBS buffer solution, ultrasonically disperse it, then add the cellulase solution, ultrasonicate, shake on a shaker overnight, centrifuge and filter, wash and dry the solid product to obtain the porous magnetic microsphere-immobilized cellulase.

[0020] Preferably, the porous magnetic microsphere-immobilized cellulase is prepared by the following method:

[0021] S2-1. Prepare porous magnetic microspheres:

[0022] Take 1.1 - 4.4 g of FeSO4·7H2O, 1.5 - 6 g of FeCl3·6H2O, and 0.75 - 3 g of polyethylene glycol, add them to 50 - 200 mL of deionized water, stir for 5 - 30 min, adjust the pH value to 9 - 10 using ammonia water, stir and react at 60 - 80 °C for 30 - 90 min, then keep warm at 70 - 95 °C for 1 - 4 h, cool, filter, and wash to obtain a precursor. Calcinate the precursor under nitrogen protection at 450 - 650 °C for 2 - 8 h to obtain porous Fe3O4 magnetic microspheres;

[0023] S2 - 2. Preparation of the immobilized carrier:

[0024] Take 2.5 - 10 g of porous Fe3O4 magnetic microspheres, add them to 100 - 400 mL of deionized water, and ultrasonically disperse for 15 - 60 min to obtain a carrier dispersion;

[0025] Add 0.96 - 3.84 g of citric acid, 1 - 4 g of chitosan with a degree of deacetylation above 70%, and 0.9 - 3.6 g of glucose to the carrier dispersion, stir for 30 - 120 min, transfer the resulting mixture to a reaction kettle, react at 170 - 200 °C for 4 - 16 h, cool, filter, wash, and dry to obtain the immobilized carrier;

[0026] S2 - 3. Loading and immobilizing cellulase:

[0027] Take 0.5 - 2 g of the immobilized carrier, add it to 25 - 100 mL of PBS buffer solution, ultrasonically disperse for 15 - 60 min, then add 2 - 20 mL of a cellulase solution with a concentration of 2.5 - 10 mg / mL, ultrasonically treat for 30 - 90 min, shake on a shaker, centrifuge, filter, wash, and dry to obtain porous magnetic microsphere - immobilized cellulase.

[0028] Preferably, in step S1, the mass ratio of the Bletilla striata powder to the deionized water is 1:10 - 100.

[0029] Preferably, in step S2, the mass ratio of the porous magnetic microsphere - immobilized cellulase to the Bletilla striata powder is 0.2 - 2:10.

[0030] Preferably, in steps S2 and S3, the ultrasonic - assisted parameters are: ultrasonic frequency 20 - 60 kHz, power 100 - 300 W.

[0031] Preferably, the preparation method of the Bletilla striata component extract for improving the pain of diabetic foot ulcers includes the following steps:

[0032] S1. Wash the Bletilla striata tuber with deionized water, air - dry, and crush it to less than 80 - 400 mesh to obtain Bletilla striata powder;

[0033] S2. Add 0.5 - 2 g of porous magnetic microsphere - immobilized cellulase into 150 - 600 g of deionized water, ultrasonically disperse for 15 - 60 min, then add 5 - 20 g of Bletilla striata powder, apply ultrasonic assistance with a frequency of 20 - 60 kHz and a power of 100 - 300 W, and stir and react at 40 - 55 °C for 2 - 8 h;

[0034] S3. Filter and collect filtrate 1. Add the filter residue into 300 g of deionized water, apply ultrasonic assistance with a frequency of 20 - 60 kHz and a power of 100 - 300 W, stir and react at 40 - 55 °C for 1 - 6 h, filter and collect filtrate 2, recycle the obtained filter residue, combine filtrate 1 and filtrate 2, concentrate at 40 - 60 °C to a total volume of 30 - 100 mL to obtain the crude Bletilla striata extract;

[0035] S4. Add 50 - 200 mL of ethanol pre - cooled to 2 - 10 °C into the crude Bletilla striata extract, stir for 0.5 - 2 h, let it stand overnight at 2 - 10 °C, centrifuge, wash the precipitate with ethanol and then redissolve it in 100 - 200 mL of deionized water, stir for 5 - 30 min, first filter the obtained solution with a micro - filter, then perform ultra - filtration, concentrate the obtained ultra - filtrate at 40 - 60 °C to a total volume of 20 - 50 mL, then add 30 - 120 mL of ethanol pre - cooled to 2 - 10 °C into the obtained concentrated solution, stir for 0.5 - 2 h, let it stand overnight at 2 - 10 °C, centrifuge, wash the precipitate with ethanol and then vacuum - dry at 20 - 30 °C for 12 - 48 h to obtain the Bletilla striata component extract.

[0036] In the second aspect of the present invention, there is provided an application of the Bletilla striata component extract as described above in the preparation of a drug for improving the pain of diabetic foot ulcers.

[0037] In the third aspect of the present invention, there is provided a drug for improving the pain of diabetic foot ulcers, comprising the Bletilla striata component extract as described above and pharmaceutically acceptable excipients; in this drug, the mass ratio of the Bletilla striata component extract is 0.1 - 50%.

[0038] In the fourth aspect of the present invention, there is provided a dressing for improving the pain of diabetic foot ulcers, which is prepared by the following method:

[0039] Mix the Bletilla striata component extract as described above with vaseline and paraffin oil, stir and heat to melt at 60 °C to obtain the dressing impregnating solution, immerse medical gauze in the dressing impregnating solution for 5 min and then take it out, perform ozone sterilization to obtain the dressing, and seal and package it;

[0040] Among them, the mass ratio of the Bletilla striata component extract: paraffin oil: vaseline is 0.1 - 10:3 - 15:100, and the mass ratio of the gauze: vaseline is 1:1 - 4.

[0041] Bletilla striata is the dried tuber of the orchid plant Bletilla striata, which is bitter, astringent, and slightly cold in nature, and has the effects of arresting bleeding with astringents, detumescence and promoting granulation,

[0042] and clearing heat and promoting diuresis. Clinically, the extract of Bletilla striata is widely used in the treatment of hemoptysis, hematemesis, ulcer bleeding, acne swelling and toxicity, etc., with remarkable curative effects, and has the good reputation of "the best in surgery". There are various active ingredients in the tuber of Bletilla striata, among which Bletilla striata polysaccharide is its main extract. The extract of Bletilla striata can significantly shorten the bleeding and clotting time, and its hemostatic effect is related to the contained colloid; the extract of Bletilla striata has an obvious protective effect on the gastric mucosa; the extract of Bletilla striata can promote granulation growth and wound healing in laboratory animal models of scalds and burns. Bletilla striata polysaccharide itself also has good antibacterial effects. In the antibacterial tests with common bacteria such as Escherichia coli and Staphylococcus aureus, researchers found that Bletilla striata polysaccharide can change the permeability of the cell wall and cell membrane of bacteria, increase its permeability, and then disrupt its cell metabolism and inhibit its growth. With the further research on the extract of Bletilla striata, it is found in the present invention that it has an obvious improvement effect on the pain symptoms of diabetic foot ulcers, and can improve and relieve pain. Based on this, the present invention applies the extract of Bletilla striata components to the treatment of diabetic foot ulcer pain, and provides a dressing for improving diabetic foot ulcer pain, which can relieve the pain of the diabetic foot ulcer wound of patients through simple external application of medicine.

[0043] The beneficial effects of the present invention are:

[0044] In the water extraction process of the present invention, porous magnetic microspheres immobilized with cellulase are added and assisted by ultrasonic action, which can decompose the cell wall of Bletilla striata, so that Bletilla striata polysaccharide can be better released, and the extraction rate of Bletilla striata polysaccharide can be improved. The ultrasonic action can promote the destruction of the cell wall of Bletilla striata and facilitate the release of Bletilla striata polysaccharide. The whole extraction process of the present invention is carried out at low temperature, which can effectively reduce the dissolution of starch and reduce the impurity content in the extract of Bletilla striata components. At the same time, since the cellulase is loaded and immobilized on the porous magnetic microspheres, the separation of cellulase from the extraction solution can be efficiently realized, avoiding the increase of protein impurity content caused by the entry of cellulase into the product of Bletilla striata component extract. Moreover, by applying a magnetic field, the porous magnetic microspheres immobilized with cellulase can be recycled and reused, and the cost can be effectively reduced.

[0045] The porous magnetic microspheres immobilized with cellulase prepared in the present invention are based on porous Fe₃O₄ magnetic microspheres, and then carbon dots are in-situ grafted on them through a hydrothermal reaction to obtain an immobilized carrier for loading and immobilizing cellulase. Finally, cellulase is loaded onto the immobilized carrier by an impregnation method to obtain the final product;

[0046] Among them, the porous Fe3O4 magnetic microspheres have a rich pore structure, which can create favorable conditions for the deposition of carbon dots during the preparation process. At the same time, they can increase the immobilization efficiency and amount of cellulase through the adsorption effect of their pores. Furthermore, they can also play a role in adsorbing impurities and improving purity during the extraction process of Bletilla striata.

[0047] Among them, the grafted carbon dots are prepared from chitosan, citric acid, and glucose as raw materials. Their surface has a rich amount of amino groups, which can react with carboxyl, hydroxyl, mercapto groups, etc. on the surface of protein molecules, and can adsorb protein molecules on the surface, thus realizing the efficient immobilization of cellulase. In addition, grafting rich carbon dots on the surface of porous Fe3O4 magnetic microspheres can increase the specific surface area and the number of active sites, which is conducive to the loading of cellulase on the surface of the immobilization carrier. In addition, the carbon dots have good permeability and can penetrate the cell wall, which is beneficial for cellulase to decompose the cell wall of Bletilla striata. At the same time, the carbon dots can also play a significant bactericidal role by destroying the bacterial cell membrane, which will have a promoting significance for the application of the Bletilla striata component extract in improving the pain of diabetic foot ulcers.

[0048] The present invention also provides the application of the Bletilla striata component extract in the treatment of diabetic foot ulcer pain. Through experiments, it is confirmed that the dressing provided by the present invention for improving diabetic foot ulcer pain can relieve the pain of the diabetic foot ulcer wound surface of patients through simple external application, and at the same time, it can also play a role in promoting the healing of the diabetic foot ulcer wound surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Test results of the extraction rate of Bletilla striata polysaccharide in the processes of the examples and the comparative examples;

[0050] Figure 2 Test results of the purity of Bletilla striata polysaccharide in the processes of the examples and the comparative examples;

[0051] Figure 3 Test results of the protein content of the products in the processes of the examples and the comparative examples;

[0052] Figure 4 Test results of the starch content of the products in the processes of the examples and the comparative examples;

[0053] Figure 5 Statistical results of the VAS score index in the application examples;

[0054] Figure 6 Test results of the effect of the dressing in promoting the healing of diabetic foot ulcer wounds in the application examples. DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention will be further described in detail below in conjunction with embodiments, so that those skilled in the art can implement it with reference to the text of the specification.

[0056] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0057] Unless otherwise specified, the test methods used in the following examples are all conventional methods. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources. For those not specified in the following examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0058] The immobilized cellulase on the porous magnetic microspheres in the following examples was all prepared by the following method:

[0059] 1. Preparation of porous magnetic microspheres:

[0060] Take 2.2 g of FeSO4·7H2O, 3 g of FeCl3·6H2O, and 1.5 g of polyethylene glycol (polyethylene glycol 2000, Beijing Solarbio Science & Technology Co., Ltd.) and add them to 100 mL of deionized water. Stir for 15 min, then adjust the pH value to 10 with 10 wt% ammonia water, stir and react at 70 °C for 45 min, then keep warm at 90 °C for 2 h. After cooling to room temperature, the product is filtered and washed with deionized water to obtain a precursor. The precursor is calcined at 500 °C for 4 h under nitrogen protection to obtain porous Fe3O4 magnetic microspheres;

[0061] 2. Preparation of immobilized carrier:

[0062] Take 5 g of porous Fe3O4 magnetic microspheres and add them to 200 mL of deionized water, and ultrasonically disperse for 30 min to obtain a carrier dispersion;

[0063] Add 1.92 g of citric acid, 2 g of chitosan (degree of deacetylation 85%, Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.), and 1.8 g of glucose to the carrier dispersion, stir for 60 min, transfer the obtained mixture to a reaction kettle, react at 190 °C for 8 h, cool to room temperature, centrifuge and filter, wash the product with deionized water, and vacuum dry at 90 °C for 12 h to obtain an immobilized carrier;

[0064] 3. Loading and immobilizing cellulase:

[0065] 1 g of immobilized carrier was added to 50 mL of PBS buffer (0.01 M, pH = 7.2), ultrasonically dispersed for 30 min, and then 10 mL of 5 mg / mL cellulase solution (Beijing Solebaugh Technology Co., Ltd.) was added. Ultrasonication was performed for 45 min, and the mixture was shaken on a shaker overnight. The mixture was centrifuged and filtered. The solid product was washed with PBS buffer and dried at 30°C to obtain porous magnetic microsphere-immobilized cellulase.

[0066] Example 1

[0067] A method for preparing a Bletilla striata extract for improving pain in diabetic foot ulcers comprises the following steps:

[0068] S1. Wash the tubers of Bletilla striata with deionized water, air-dry them, and then grind them into a size of less than 200 mesh to obtain Bletilla striata powder;

[0069] S2. Add 1 g of porous magnetic microsphere-immobilized cellulase to 300 g of deionized water and ultrasonically disperse for 30 min. Then, add 10 g of Bletilla striata powder and apply ultrasonic assistance at a frequency of 40 kHz and a power of 200 W. Stir and react at 45°C for 4 h.

[0070] S3, filtering and collecting filtrate 1, adding the filter residue to 300 g of deionized water, stirring and reacting at 45°C for 3 h with the aid of ultrasound, filtering and collecting filtrate 2, recovering the obtained filter residue, combining filtrate 1 and filtrate 2, and concentrating at 45°C to a total volume of 50 mL to obtain a crude extract of Bletilla striata;

[0071] S4. Add 100 mL of ethanol precooled to 4°C to the crude extract of Bletilla striata, stir for 1 h, let stand at 4°C overnight, centrifuge, wash the precipitate with ethanol and redissolve it in 150 mL of deionized water, stir for 15 min, and first filter the resulting solution with a microfilter (0.22 μm). Ultrafiltration is performed on the filtrate at 45°C with an ultrafiltration pressure of 0.2 MPa, an ultrafiltration molecular weight cutoff of 50 KDa, and a filter ratio of 3:7. Concentrate the ultrafiltrate at 45°C to a total volume of 30 mL, then add 70 mL of ethanol precooled to 4°C to the concentrated solution, stir for 1 h, let stand at 4°C overnight, centrifuge, wash the precipitate with ethanol and vacuum dry it at 25°C for 24 h to obtain a Bletilla striata component extract.

[0072] Example 2

[0073] A method for preparing a Bletilla striata extract for improving pain in diabetic foot ulcers comprises the following steps:

[0074] S1. Wash the tubers of Bletilla striata with deionized water, air-dry them, and then grind them into a size of less than 200 mesh to obtain Bletilla striata powder;

[0075] S2. Add 1 g of porous magnetic microsphere-immobilized cellulase to 400 g of deionized water, ultrasonically disperse for 30 min, then add 10 g of Bletilla striata powder, apply ultrasonic assistance at a frequency of 40 kHz and a power of 200 W, and stir and react at 45 °C for 5 h;

[0076] S3. Filter and collect filtrate 1. Add the filter residue to 400 g of deionized water again, carry out ultrasonic assistance and stir and react at 45 °C for 3 h, filter and collect filtrate 2. Recover the obtained filter residue, combine filtrate 1 and filtrate 2, and concentrate to a total volume of 50 mL at 45 °C to obtain a crude Bletilla striata extract;

[0077] S4. Add 100 mL of ethanol pre-cooled to 4 °C to the crude Bletilla striata extract, stir for 1 h, let stand overnight at 4 °C, centrifuge. Wash the precipitate with ethanol and then redissolve it in 150 mL of deionized water, stir for 15 min. First filter the obtained solution with a microfiltration instrument (0.22 μm), ultrafilter the obtained filtrate at 45 °C, the ultrafiltration pressure is 0.2 MPa, the ultrafiltration cut-off molecular weight is 50 KDa, and the filtration cut-off ratio is 3:7. Concentrate the obtained ultrafiltrate to a total volume of 30 mL at 45 °C, then add 70 mL of ethanol pre-cooled to 4 °C to the obtained concentrated solution, stir for 1 h, let stand overnight at 4 °C, centrifuge. Wash the precipitate with ethanol and then vacuum dry at 25 °C for 24 h to obtain an extract of Bletilla striata components.

[0078] Example 3

[0079] A preparation method of an extract of Bletilla striata components for improving the pain of diabetic foot ulcers, comprising the following steps:

[0080] S1. Wash the Bletilla striata tubers with deionized water, air-dry and then crush them to less than 200 meshes to obtain Bletilla striata powder;

[0081] S2. Add 1 g of porous magnetic microsphere-immobilized cellulase to 300 g of deionized water, ultrasonically disperse for 30 min, then add 10 g of Bletilla striata powder, apply ultrasonic assistance at a frequency of 40 kHz and a power of 250 W, and stir and react at 50 °C for 4 h;

[0082] S3. Filter and collect filtrate 1. Add the filter residue to 300 g of deionized water again, carry out ultrasonic assistance and stir and react at 50 °C for 3 h, filter and collect filtrate 2. Recover the obtained filter residue, combine filtrate 1 and filtrate 2, and concentrate to a total volume of 50 mL at 45 °C to obtain a crude Bletilla striata extract;

[0083] S4. Add 100 mL of ethanol pre-cooled to 4 °C to the crude Bletilla striata extract, stir for 1 h, let it stand overnight at 4 °C, centrifuge, wash the precipitate with ethanol and then redissolve it in 150 mL of deionized water, stir for 15 min, first filter the obtained solution with a microfiltration instrument (0.22 μm), ultrafilter the obtained filtrate at 45 °C, with an ultrafiltration pressure of 0.2 MPa, an ultrafiltration cut-off molecular weight of 50 KDa, and a filtration cut-off ratio of 3:7. Concentrate the obtained ultrafiltrate at 45 °C to a total volume of 30 mL, then add 70 mL of ethanol pre-cooled to 4 °C to the obtained concentrated solution, stir for 1 h, let it stand overnight at 4 °C, centrifuge, wash the precipitate with ethanol and then vacuum dry it at 25 °C for 24 h to obtain the Bletilla striata component extract.

[0084] Example 4

[0085] A preparation method of a Bletilla striata component extract for improving the pain of diabetic foot ulcers, comprising the following steps:

[0086] S1. Wash the Bletilla striata tubers with deionized water, air-dry them and then crush them to less than 200 mesh to obtain Bletilla striata powder;

[0087] S2. Add 1 g of porous magnetic microsphere-immobilized cellulase to 400 g of deionized water, ultrasonically disperse for 30 min, then add 10 g of Bletilla striata powder, apply ultrasonic assistance with a frequency of 40 kHz and a power of 200 W, and stir and react at 45 °C for 5 h;

[0088] S3. Filter and collect filtrate 1, add the filter residue to 200 g of deionized water again, perform ultrasonic assistance and stir and react at 45 °C for 2 h, filter and collect filtrate 2, recycle the obtained filter residue, combine filtrate 1 and filtrate 2, and concentrate to a total volume of 50 mL at 45 °C to obtain the crude Bletilla striata extract;

[0089] S4. Add 100 mL of ethanol pre-cooled to 4 °C to the crude Bletilla striata extract, stir for 1 h, let it stand overnight at 4 °C, centrifuge, wash the precipitate with ethanol and then redissolve it in 150 mL of deionized water, stir for 15 min, first filter the obtained solution with a microfiltration instrument (0.22 μm), ultrafilter the obtained filtrate at 45 °C, with an ultrafiltration pressure of 0.2 MPa, an ultrafiltration cut-off molecular weight of 50 KDa, and a filtration cut-off ratio of 3:7. Concentrate the obtained ultrafiltrate at 45 °C to a total volume of 30 mL, then add 70 mL of ethanol pre-cooled to 4 °C to the obtained concentrated solution, stir for 1 h, let it stand overnight at 4 °C, centrifuge, wash the precipitate with ethanol and then vacuum dry it at 25 °C for 24 h to obtain the Bletilla striata component extract.

[0090] Comparative Example 1

[0091] A preparation method of a Bletilla striata component extract for improving the pain of diabetic foot ulcers, comprising the following steps:

[0092] S1. Wash the Bletilla striata tuber with deionized water, air-dry it, and then crush it to less than 200 mesh to obtain Bletilla striata powder;

[0093] S2. Add 10 g of Bletilla striata powder to 300 g of deionized water, apply ultrasonic assistance with a frequency of 40 kHz and a power of 200 W, and stir and react at 45 °C for 4 h;

[0094] S3. Filter and collect filtrate 1. Add the filter residue to 300 g of deionized water again, stir and react under ultrasonic assistance at 45 °C for 3 h, filter and collect filtrate 2. The obtained filter residue is recycled. Combine filtrate 1 and filtrate 2, and concentrate to a total volume of 50 mL at 45 °C to obtain the crude Bletilla striata extract;

[0095] S4. Add 100 mL of ethanol pre-cooled to 4 °C to the crude Bletilla striata extract, stir for 1 h, let it stand overnight at 4 °C, centrifuge. Wash the precipitate with ethanol and then redissolve it in 150 mL of deionized water, stir for 15 min. First, filter the obtained solution with a microfiltration instrument (0.22 μm). Ultrafilter the obtained filtrate at 45 °C with an ultrafiltration pressure of 0.2 MPa and an ultrafiltration cut-off molecular weight of 50 KDa, and the filtration cut-off ratio is 3:7. Concentrate the obtained ultrafiltrate to a total volume of 30 mL at 45 °C. Then add 70 mL of ethanol pre-cooled to 4 °C to the obtained concentrated solution, stir for 1 h, let it stand overnight at 4 °C, centrifuge. Wash the precipitate with ethanol and then vacuum dry it at 25 °C for 24 h to obtain the Bletilla striata component extract.

[0096] Comparative Example 2

[0097] The difference between this example and Example 1 is only that:

[0098] The porous magnetic microsphere-immobilized cellulase in this example is prepared by the following method:

[0099] 1. Prepare porous magnetic microspheres:

[0100] Take 2.2 g of FeSO4·7H2O, 3 g of FeCl3·6H2O, and 1.5 g of polyethylene glycol (polyethylene glycol 2000, Beijing Solarbio Science & Technology Co., Ltd.), add them to 100 mL of deionized water, stir for 15 min, then adjust the pH value to 10 with 10 wt% ammonia water, stir and react at 70 °C for 45 min, then keep it at 90 °C for 2 h. After cooling to room temperature, filter and wash the product with deionized water to obtain the precursor. Calcinate the precursor under nitrogen protection at 500 °C for 4 h to obtain porous Fe3O4 magnetic microspheres as the immobilization carrier;

[0101] 2. Load and immobilize cellulase:

[0102] Take 1 g of the immobilized carrier and add it to 50 mL of PBS buffer (0.01 M, pH = 7.2). Ultrasonically disperse for 30 min, then add 10 mL of a cellulase solution with a concentration of 5 mg / mL (Beijing Solarbio Science & Technology Co., Ltd.). Ultrasonicate for 45 min, shake overnight on a shaker, centrifuge and filter. Wash the solid product with PBS buffer and dry at 30 °C to obtain porous magnetic microsphere-immobilized cellulase.

[0103] Comparative Example 3

[0104] The difference between this example and Example 1 is only that:

[0105] The porous magnetic microsphere-immobilized cellulase in this example was prepared by the following method:

[0106] 1. Preparation of porous magnetic microspheres:

[0107] Take 2.2 g of FeSO4·7H2O and 3 g of FeCl3·6H2O and add them to 100 mL of deionized water. Stir for 15 min, then adjust the pH value to 10 using 10 wt% ammonia water. Stir and react at 70 °C for 45 min, then keep warm at 90 °C for 2 h. After cooling to room temperature, filter and wash the product with deionized water to obtain a precursor. Calcinate the precursor at 500 °C for 4 h under nitrogen protection to obtain porous Fe3O4 magnetic microspheres;

[0108] 2. Preparation of the immobilized carrier:

[0109] Take 5 g of porous Fe3O4 magnetic microspheres and add them to 200 mL of deionized water. Ultrasonically disperse for 30 min to obtain a carrier dispersion;

[0110] Add 1.92 g of citric acid, 2 g of chitosan (degree of deacetylation 85%, Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.) and 1.8 g of glucose to the carrier dispersion. Stir for 60 min, transfer the resulting mixture to a reaction kettle, react at 190 °C for 8 h, cool to room temperature, centrifuge and filter. Wash the product with deionized water and dry it under vacuum at 90 °C for 12 h to obtain the immobilized carrier;

[0111] 3. Loading and immobilizing cellulase:

[0112] Take 1 g of the immobilized carrier and add it to 50 mL of PBS buffer (0.01 M, pH = 7.2). Ultrasonically disperse for 30 min, then add 10 mL of a cellulase solution with a concentration of 5 mg / mL (Beijing Solarbio Science & Technology Co., Ltd.). Ultrasonicate for 45 min, shake overnight on a shaker, centrifuge and filter. Wash the solid product with PBS buffer and dry at 30 °C to obtain porous magnetic microsphere-immobilized cellulase.

[0113] Comparative Example 4

[0114] A preparation method of a Bletilla striata component extract for improving the pain of diabetic foot ulcers, comprising the following steps:

[0115] S1. Wash the Bletilla striata tubers with deionized water, air-dry them, and then crush them to less than 200 mesh to obtain Bletilla striata powder;

[0116] S2. Add 10 g of Bletilla striata powder to 300 g of deionized water, apply ultrasonic assistance with a frequency of 40 kHz and a power of 200 W, and stir and react at 85 °C for 4 h;

[0117] S3. Filter and collect filtrate 1. Add the filter residue to 300 g of deionized water again, perform ultrasonic assistance, stir and react at 85 °C for 3 h, filter and collect filtrate 2, recycle the obtained filter residue, combine filtrate 1 and filtrate 2, and concentrate to a total volume of 50 mL at 45 °C to obtain a crude Bletilla striata extract;

[0118] S4. Add 100 mL of ethanol pre-cooled to 4 °C to the crude Bletilla striata extract, stir for 1 h, let it stand overnight at 4 °C, centrifuge, wash the precipitate with ethanol and then redissolve it in 150 mL of deionized water, stir for 15 min, first filter the obtained solution with a microfilter (0.22 μm), ultrafilter the obtained filtrate at 45 °C, the ultrafiltration pressure is 0.2 MPa, the ultrafiltration cut-off molecular weight is 50 KDa, and the filtration ratio is 3:7. Concentrate the obtained ultrafiltrate to a total volume of 30 mL at 45 °C, then add 70 mL of ethanol pre-cooled to 4 °C to the obtained concentrated solution, stir for 1 h, let it stand overnight at 4 °C, centrifuge, wash the precipitate with ethanol and then vacuum dry it at 25 °C for 24 h to obtain the Bletilla striata component extract.

[0119] Comparative Example 5

[0120] A preparation method of a Bletilla striata component extract for improving the pain of diabetic foot ulcers, comprising the following steps:

[0121] S1. Wash the Bletilla striata tubers with deionized water, air-dry them, and then crush them to less than 200 mesh to obtain Bletilla striata powder;

[0122] S2. Add 10 g of Bletilla striata powder to 300 g of deionized water, apply ultrasonic assistance with a frequency of 40 kHz and a power of 200 W, and stir and react at 95 °C for 4 h;

[0123] S3. Filter and collect filtrate 1. Add the filter residue to 300 g of deionized water again, perform ultrasonic assistance, stir and react at 95 °C for 3 h, filter and collect filtrate 2, recycle the obtained filter residue, combine filtrate 1 and filtrate 2, and concentrate to a total volume of 50 mL at 45 °C to obtain a crude Bletilla striata extract;

[0124] S4. Add 100 mL of ethanol pre-cooled to 4°C to the crude extract of Bletilla striata, stir for 1 h, let it stand overnight at 4°C, centrifuge, wash the precipitate with ethanol and then redissolve it in 150 mL of deionized water, stir for 15 min. First, filter the obtained solution with a microfiltration instrument (0.22 μm), ultrafilter the obtained filtrate at 45°C with an ultrafiltration pressure of 0.2 MPa, an ultrafiltration cut-off molecular weight of 50 KDa, and a filtration cut-off ratio of 3:7. Concentrate the obtained ultrafiltrate at 45°C to a total volume of 30 mL, then add 70 mL of ethanol pre-cooled to 4°C to the obtained concentrated solution, stir for 1 h, let it stand overnight at 4°C, centrifuge, wash the precipitate with ethanol and then vacuum dry it at 25°C for 24 h to obtain the Bletilla striata component extract.

[0125] Process index test

[0126] Measure the extraction rate of Bletilla striata polysaccharide, the purity of Bletilla striata polysaccharide, and the starch and protein contents in the extract in the processes of the examples and the comparative examples; among them, the extraction rate of crude polysaccharide (%) = dry weight of the extract (g) / powder of Bletilla striata tubers (g).

[0127] The measurement results are shown in Table 1 below:

[0128] Table 1

[0129]

[0130] It can be seen from the test results that the extraction rates in Examples 1 - 4 are high, the product purity is high, there are few protein impurities, and no starch appears in the products; in Comparative Example 1, no porous magnetic microsphere-immobilized cellulase was added during the extraction process, and the extraction rate was too low; in Comparative Example 2, no carbon dots were loaded on the immobilized carrier, resulting in a decrease in the cellulase loading amount, a decrease in the specific surface area, and a decrease in the cell wall decomposition efficiency, so the extraction rate and purity index decreased; in Comparative Example 3, the porosity of the magnetic microspheres decreased, resulting in a decrease in the cellulase loading amount and a decrease in the extraction rate and purity index; in Comparative Examples 4 and 5, a high-temperature extraction process was used and no porous magnetic microsphere-immobilized cellulase was added, and a large amount of starch impurities appeared in the products.

[0131] Application example

[0132] A dressing for improving the pain of diabetic foot ulcers is prepared by the following method:

[0133] Mix the Bletilla striata component extract prepared in Example 1 with vaseline (Tianjin Jindong Tianzheng Fine Chemical Reagent Factory) and paraffin oil (melting point 58°C, Hebei Xinwanbang High Polymer Materials Technology Co., Ltd.), stir and heat to 60°C to melt to obtain a dressing impregnating solution. Immerse medical gauze in the dressing impregnating solution for 5 min and then take it out, sterilize it with ozone to obtain the dressing, and package it in a sealed manner;

[0134] The mass ratio of the Bletilla striata extract, paraffin oil, and vaseline is 4:6:100, and the mass ratio of gauze to vaseline is 1:2.

[0135] I. Application effect test of the dressing in improving the pain of diabetic foot ulcers:

[0136] 1. Subjects: 60 volunteers were selected from inpatients at the Chinese PLA General Hospital. The volunteers were aged 30 - 60 years, all were diabetic patients with symptoms of foot ulcers, and their body mass index (BMI) was 18 to 30 kg / m 2 , including 37 males and 23 females.

[0137] 2. Medication method:

[0138] After rinsing the wound surface with 0.9% normal saline, apply the dressing prepared by the above method to the wound surface, fix it with sterile gauze, and remove the dressing after 2 hours.

[0139] 3. Effect evaluation method:

[0140] (1) Use the VAS pain scale to quantify the pain scores of patients before and after medication to evaluate the effect of the dressing in relieving pain.

[0141] (2) The VAS pain scale, namely the visual analogue scale, is a commonly used pain assessment tool for clinical patients. Scoring method: Draw a 10 - cm horizontal line on paper. One end of the line is 0, indicating no pain; the other end is 10, indicating severe pain. Patients mark the corresponding position on the horizontal line according to the pain level they feel, measure the distance from the starting point to the marked point, and the obtained value is the pain score.

[0142] (3) Scoring criteria: 0 points indicates no pain; 1 - 3 points indicates mild pain, which patients can tolerate and does not affect sleep; 4 - 6 points indicates moderate pain, with obvious pain that affects sleep but is still tolerable; 7 - 10 points indicates severe pain, with intense pain that cannot be tolerated, seriously affecting sleep, and may be accompanied by symptoms such as sweating, dizziness, and fatigue. When using the VAS pain scale, the researcher needs to first explain the scoring method clearly to the patient to ensure that the patient understands and can accurately express their pain level. This method can more intuitively reflect the patient's pain perception and is a commonly used tool for quantifying pain intensity on a visual scale.

[0143] (4) Evaluation results: The pain score results before and after medication are shown in Tables 2 and 3 below. It can be seen that after using the dressing, the pain score decreased significantly, indicating that the dressing has the effect of improving the pain of diabetic foot ulcers.

[0144] Table 2 VAS score and number of people statistics

[0145] VAS score / score Number of patients before drug administration Number of patients after drug administration 3 0 11 4 0 5 5 0 14 6 9 30 7 29 0 8 12 0 9 10 0

[0146] Table 3 Statistical Index of VAS Score

[0147] Average VAS score Min(VAS score) MaX(VAS score) Before drug administration 7.38 6 9 After drug administration 5.05 3 6

[0148] Among them, Min and Max respectively represent the minimum and maximum values of the VAS score.

[0149] II. Test on the Effect of the Dressing in Promoting the Healing of Diabetic Foot Ulcer Wounds:

[0150] (1) Subject's condition: Male, 68 years old, diabetic foot;

[0151] (2) Medication administration method:

[0152] After rinsing the wound with 0.9% normal saline, apply the dressing prepared by the above method to the wound, fix it with sterile gauze, and remove the dressing after 2 hours.

[0153] Refer to Figure 6 For the healing effect of the diabetic foot ulcer wound after the patient has taken the medicine for different days, it can be seen that this dressing can promote the healing of the diabetic foot ulcer wound.

[0154] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the description and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. A preparation method of a Bletilla striata component extract for improving the pain of diabetic foot ulcers, characterized in that, It includes the following steps: S1. Wash the tubers of Bletilla striata with deionized water, air-dry them and then crush to obtain Bletilla striata powder; S2. Add the porous magnetic microsphere-immobilized cellulase into deionized water, ultrasonically disperse it, then add the Bletilla striata powder, and stir and react under ultrasonic assistance and heating; S3. Filter and collect filtrate 1. Add the filter residue into deionized water again, stir and react under ultrasonic assistance and heating, filter and collect filtrate 2, combine filtrate 1 and filtrate 2, concentrate to obtain the crude Bletilla striata extract; S4. Add ethanol to the crude Bletilla striata extract, stir, let stand overnight, centrifuge, wash the precipitate with ethanol and then redissolve it in deionized water, stir, first filter the obtained solution with a microfiltration instrument, then perform ultrafiltration, concentrate the obtained ultrafiltrate, then add ethanol, stir, let stand overnight, centrifuge, wash the precipitate with ethanol and then dry to obtain the Bletilla striata component extract.

2. The preparation method of the Bletilla striata component extract for improving the pain of diabetic foot ulcers according to claim 1, characterized in that, The porous magnetic microsphere-immobilized cellulase is prepared by the following method: S2-1. Prepare porous magnetic microspheres: Take FeSO4·7H2O, FeCl3·6H2O, and polyethylene glycol, add them into deionized water, stir, then adjust the pH value to alkaline with ammonia water, stir and react under heating, then heat and keep warm, cool and wash, and calcine the obtained precursor to obtain porous Fe3O4 magnetic microspheres; S2-2. Prepare the immobilized carrier: Take the porous Fe3O4 magnetic microspheres and add them into deionized water, ultrasonically disperse to obtain a carrier dispersion; Add citric acid, chitosan, and glucose to the carrier dispersion, stir, transfer the obtained mixture to a reaction kettle, react under heating, cool, filter, wash, and dry after the reaction to obtain the immobilized carrier; S2-3. Load and immobilize cellulase: Take the immobilized carrier, add it into PBS buffer solution, ultrasonically disperse, then add the cellulase solution, ultrasonicate, shake on a shaker overnight, centrifuge and filter, wash and dry the solid product to obtain the porous magnetic microsphere-immobilized cellulase.

3. The preparation method of the Bletilla striata component extract for improving the pain of diabetic foot ulcers according to claim 2, characterized in that, The porous magnetic microsphere-immobilized cellulase is prepared by the following method: S2-1. Prepare porous magnetic microspheres: Take 1.1 - 4.4 g of FeSO4·7H2O, 1.5 - 6 g of FeCl3·6H2O, 0.75 - 3 g of polyethylene glycol, add them into 50 - 200 mL of deionized water, stir for 5 - 30 min, use ammonia water to adjust the pH value to 9 - 10, stir and react at 60 - 80 °C for 30 - 90 min, then keep warm at 70 - 95 °C for 1 - 4 h, cool and then filter and wash to obtain a precursor, and calcine the precursor under nitrogen protection at 450 - 650 °C for 2 - 8 h to obtain porous Fe3O4 magnetic microspheres; S2-2. Prepare the immobilized carrier: Take 2.5 - 10 g of porous Fe3O4 magnetic microspheres and add them into 100 - 400 mL of deionized water, ultrasonically disperse for 15 - 60 min to obtain a carrier dispersion; Add 0.96 - 3.84 g of citric acid, 1 - 4 g of chitosan with a degree of deacetylation above 70%, and 0.9 - 3.6 g of glucose to the carrier dispersion liquid, stir for 30 - 120 min, transfer the obtained mixture to a reaction kettle, react at 170 - 200 °C for 4 - 16 h, cool, filter, wash, and dry to obtain the immobilized carrier; S2 - 3. Load and immobilize cellulase: Take 0.5 - 2 g of the immobilized carrier and add it to 25 - 100 mL of PBS buffer solution, ultrasonically disperse for 15 - 60 min, then add 2 - 20 mL of a cellulase solution with a concentration of 2.5 - 10 mg / mL, ultrasonically for 30 - 90 min, shake on a shaker, centrifuge, filter, wash, and dry to obtain the porous magnetic microsphere - immobilized cellulase.

4. The preparation method of the bletilla striata component extract for improving the pain of diabetic foot ulcers according to claim 1, characterized in that, In step S1, the mass ratio of the Bletilla striata powder to deionized water is 1:10 - 100.

5. The preparation method of the bletilla striata component extract for improving the pain of diabetic foot ulcers according to claim 1, characterized in that, In step S2, the mass ratio of the porous magnetic microsphere - immobilized cellulase to the Bletilla striata powder is 0.2 - 2:

10.

6. The preparation method of the Bletilla striata component extract for improving the pain of diabetic foot ulcers according to claim 1, characterized in that, In steps S2 and S3, the ultrasonic assistance parameters are: ultrasonic frequency 20 - 60 kHz, power 100 - 300 W.

7. The preparation method of the bletilla striata component extract for improving the pain of diabetic foot ulcers according to claim 1, characterized in that, It includes the following steps: S1. Wash the Bletilla striata tubers with deionized water, air - dry, and then crush them to below 80 - 400 mesh to obtain the Bletilla striata powder; S2. Add 0.5 - 2 g of the porous magnetic microsphere - immobilized cellulase to 150 - 600 g of deionized water, ultrasonically disperse for 15 - 60 min, then add 5 - 20 g of the Bletilla striata powder, apply ultrasonic assistance with a frequency of 20 - 60 kHz and a power of 100 - 300 W, and stir and react at 40 - 55 °C for 2 - 8 h; S3. Filter and collect filtrate 1, add the filter residue to 300 g of deionized water again, apply ultrasonic assistance with a frequency of 20 - 60 kHz and a power of 100 - 300 W, stir and react at 40 - 55 °C for 1 - 6 h, filter and collect filtrate 2, recycle the obtained filter residue, combine filtrate 1 and filtrate 2, concentrate at 40 - 60 °C to a total volume of 30 - 100 mL to obtain the crude Bletilla striata extract; S4. Add 50 - 200 mL of ethanol pre - cooled to 2 - 10 °C to the crude Bletilla striata extract, stir for 0.5 - 2 h, let it stand overnight at 2 - 10 °C, centrifuge, wash the precipitate with ethanol and then redissolve it in 100 - 200 mL of deionized water, stir for 5 - 30 min, first filter the obtained solution with a microfiltration instrument, then perform ultrafiltration, concentrate the obtained ultrafiltrate at 40 - 60 °C to a total volume of 20 - 50 mL, then add 30 - 120 mL of ethanol pre - cooled to 2 - 10 °C to the obtained concentrated solution, stir for 0.5 - 2 h, let it stand overnight at 2 - 10 °C, centrifuge, wash the precipitate with ethanol and then vacuum - dry at 20 - 30 °C for 12 - 48 h to obtain the Bletilla striata component extract.

8. Use of the Bletilla striata component extract as described in any one of claims 1 - 7 in the preparation of a drug for improving the pain of diabetic foot ulcers.

9. A drug for improving the pain of diabetic foot ulcers, comprising the extract of the Bletilla striata component described in any one of claims 1-7 and pharmaceutically acceptable excipients; in this drug, the mass ratio of the extract of the Bletilla striata component is 0.1-50%.

10. A dressing for improving the pain of diabetic foot ulcers, characterized in that, It is prepared by the following method: Mix the extract of the Bletilla striata component described in any one of claims 1-7 with petrolatum and paraffin oil, stir and heat to 60°C to melt to obtain a dressing impregnating solution, immerse medical gauze in the dressing impregnating solution for 5 minutes and then take it out, and obtain the dressing after ozone sterilization, and seal and package it; Among them, the mass ratio of the extract of the Bletilla striata component: paraffin oil: petrolatum is 0.1-10: 3-15: 100, and the mass ratio of gauze: petrolatum is 1: 1-4.

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