Effect of sugar foot health cream in promoting wound healing of sugar foot ulcer

Abstract

The invention provides an effect of a sugar foot health cream in promoting wound healing of sugar foot ulcer, relates to the technical field of traditional Chinese medicine components, and is technically characterized in that the sugar foot health cream is used for promoting wound healing of sugar foot ulcer and comprises the following traditional Chinese medicine components: rhizoma atractylodis, cortex phellodendri, semen coicis, cortex cinnamomi, radix angelicae sinensis, radix astragali and borneol. According to the application, a rat lower limb ischemic type 1 DFU model is constructed, and the model verifies that the Gangsukang ointment provided by the application is suitable for various DFU dialectical types; and by improving the dosage form, the preparation can be directly applied to the wound surface, so that the curative effect is improved, the stimulation to the wound surface is reduced, and the compliance of a patient is enhanced. Cream is a multi-component traditional Chinese medicine and can improve the anti-inflammatory effect; wound tissue regeneration is promoted; the ulcer surface healing is accelerated.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine formula, and particularly to the role of Tangzukang Cream in promoting the healing of diabetic foot ulcer wounds. Background Art

[0002] Diabetic Foot Ulcer (DFU), as one of the most serious chronic complications of diabetes, has become an important challenge in the global public health field. According to the statistics of the International Diabetes Federation (IDF), about 20% of global diabetic patients will develop DFU during their disease course, and about 25% of these cases will eventually develop into amputation, resulting in a sharp decline in the quality of life of patients and a heavy medical burden. This pathological state characterized by foot tissue necrosis, ulcer formation and non-healing is essentially a local outbreak of multi-system damage caused by diabetic metabolic disorders.

[0003] Current clinical treatments mainly adopt stepwise comprehensive interventions, including intensive blood glucose control, mechanical decompression, surgical debridement, anti-infection treatment and the application of wound dressings. However, these traditional methods have significant limitations: systematic blood glucose management is difficult to reverse the already formed tissue damage, surgical debridement may damage the viable tissue at the wound edge, the problem of antibiotic resistance is becoming increasingly severe, and traditional dressings can only provide passive protection and lack active repair functions. More intractably, the microenvironment of DFU wounds has high heterogeneity, with significant differences in the degree of ischemia, type of infection, range of tissue necrosis, etc. among different patients, resulting in the ineffectiveness of standardized treatment regimens. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem of the lack of targeted treatment regimens in the prior art.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A Tangzukang Cream for promoting the healing of diabetic foot ulcer wounds, the cream comprising the following traditional Chinese medicine components: Atractylodes lancea, Phellodendron amurense, Coix lacryma-jobi, Cinnamomum cassia, Angelica sinensis, Astragalus membranaceus, Borneol.

[0007] Preferably, the mass ratio of the traditional Chinese medicine components is as follows: Atractylodes lancea 200 parts, Phellodendron amurense 200 parts, Coix lacryma-jobi 600 parts, Cinnamomum cassia 60 parts, Angelica sinensis 300 parts, Astragalus membranaceus 300 parts, Borneol 4 parts.

[0008] Preferably, the cream further comprises materials for preparing the oil phase: Stearic Acid, Glyceryl Monostearate, Light Liquid Paraffin, Ethylparaben, Polysorbate 80, Sorbitan Oleate, Benzoic Acid.

[0009] Preferably, the cream further comprises glycerol and purified water.

[0010] Preferably, the preparation process of the cream is as follows:

[0011] S1: Weigh the six herbs of Atractylodes lancea, Phellodendron amurense, Coix lacryma-jobi, Cinnamomum cassia, Angelica sinensis, and Astragalus membranaceus according to the formula amount, add 8 times the amount of water, soak, decoct, and filter; add 6 times the amount of water to the filter residue, decoct, and filter, and combine the two filtrates; concentrate the filtrate into an extract, set aside, dry, pulverize, and set aside;

[0012] S2: Prepare the oil phase;

[0013] S3: Place the extract in S1 and purified water in a container, heat, add glycerol while stirring, and let stand to obtain the water phase

[0014] S4: Add the water phase in S3 to the oil phase in S2, and mix evenly to obtain Tangzukang Ointment.

[0015] Preferably, weigh stearic acid, glycerol monostearate, light liquid paraffin, ethylparaben, polysorbate 80, sorbitan oleate, benzoic acid, and borneol. After weighing, place the above materials in a dry container, stir, and heat to 78 °C - 82 °C until completely dissolved, then let stand and set aside.

[0016] Preferably, the heating temperature in S3 is 78 °C - 82 °C.

[0017] This application also provides the use of the above-mentioned Tangzukang Ointment in the preparation of a drug for promoting the healing of diabetic foot ulcer wounds.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] In this application, a rat model of ischemic type 1 diabetic foot ulcer (DFU) was constructed, and through the above model, it was verified that the Tangzukang Ointment provided in this application is applicable to various DFU dialectical types, especially ischemic diabetic foot ulcers; and through dosage form improvement, it can be directly administered to the wound surface, increasing the efficacy, reducing the irritation to the wound surface, and enhancing the compliance of patients. The cream is a multi-component traditional Chinese medicine: it improves the anti-inflammatory effect; promotes wound tissue regeneration; and accelerates the healing of ulcer surfaces. Brief Description of the Drawings

[0020] Figure 1 is the Tangzukang Cream prepared in one embodiment of the present invention;

[0021] Figure 2 is the statistics of the healing of the rat foot ulcer wound surface and the ulcer healing area in one embodiment of the present invention. Among them, A: Representative photos of the wound surface healing of each group; B. Statistical chart of the wound surface healing rate of each group. *P < 0.05, ****P < 0.0001

[0022] Figure 3Statistics on the significant promotion of wound re-epithelialization by Tangzukang Cream in an embodiment of the present invention. Among them, A: Representative HE staining images of wounds in each group at 14 days and 21 days; B: Statistical chart of wound re-epithelialization in each group. **P<0.01, ***P<0.001.

[0023] Figure 4 Masson staining and collagen content statistics of wound tissues in an embodiment of the present invention. Among them, A: Representative Masson staining images of wounds in each group at 14 days and 21 days; B: Statistical chart of wound collagen content in each group. **P<0.01, ****P<0.0001.

[0024] Figure 5 Expression levels of pro-inflammatory factors in wound tissues in an embodiment of the present invention. Among them, A - C: qPCR detection of the expression of inflammatory factors IL-1β, IL-6, and TNF-α. **P<0.01. Specific Embodiments

[0025] The present invention will be further described in detail below in conjunction with specific embodiments.

[0026] A kind of Tangzukang Cream for promoting the healing of diabetic foot ulcer wounds. The cream comprises the following weight parts calculated by dry weight after processing: Atractylodes lancea 200 parts, Phellodendron amurense 200 parts, Coix lacryma-jobi 600 parts, Cinnamon 60 parts, Angelica sinensis 300 parts, Astragalus membranaceus 300 parts, Borneol 4 parts, Glycerol 76 parts, Stearic acid 76 parts, Monoglyceride (Glycerol monostearate) 76 parts, Light liquid paraffin (0.851) 190 parts, Tween - 80 (Polysorbate 80) 38 parts, Span - 80 (Sorbitan oleate) 19 parts, Ethylparaben 4 parts, Benzoic acid 8 parts, and Purified water 513 parts.

[0027] The preparation method of the above-mentioned Tangzukang Ointment is as follows:

[0028] S1: Preparation of extract:

[0029] Weigh Atractylodes lancea, Phellodendron amurense, Coix lacryma-jobi, Cinnamon, Angelica sinensis, and Astragalus membranaceus according to the formula, add 8 times the amount of water, soak for 30 minutes, decoct for 2 hours, and filter; add 6 times the amount of water to the filter residue, decoct for 1 hour, filter, and combine the two filtrates; concentrate the filtrate to an extract with a relative density of about 1.20 - 1.25 (80 °C), set aside, dry, pulverize, and set aside;

[0030] S2: Preparation of oil phase:

[0031] Weigh stearic acid, glycerol monostearate, light liquid paraffin, ethylparaben, polysorbate - 80, sorbitan oleate, benzoic acid, and borneol according to the above formula. After weighing, place the above materials in a dry container, stir, and heat to 78 °C - 82 °C until completely melted, let stand, and set aside;

[0032] S3: Preparation of aqueous phase:

[0033] Place the extract obtained in S1 and purified water in accordance with the formula ratio in a container, heat to 78°C - 82°C, add glycerol while stirring, and let it stand for later use.

[0034] S4: Preparation of combined phase:

[0035] Slowly add the aqueous phase obtained in S3 to the oil phase, mix evenly, and dispense to obtain Tangzukang Ointment.

[0036] In addition, the present application also provides the use of the above-mentioned Tangzukang Ointment in the preparation of a drug for promoting the healing of diabetic foot ulcer wounds.

[0037] The above content is elaborated below in combination with specific verification experiments:

[0038] Experimental materials and sources:

[0039]

[0040] Example 1: Establishment and sampling of a model of ischemic foot ulcer in T1DM rats

[0041] The grouping of the animal experiment is as follows: (1) Control group of foot ulcer without diabetes (Control); (2) Model group (DFU); (3) DFU + HUVECs-Exos treatment group (Exos).

[0042] (1) Preparation of STZ-sodium citrate buffer

[0043] Preparation of solution A and solution B: Weigh 1.05 g of citric acid with a balance and add it to 50 mL of ddH2O to prepare solution A; weigh 1.47 g of sodium citrate and add it to 50 mL of ddH2O to prepare solution B, and store at 4°C. After complete dissolution, mix solution A and solution B in a volume ratio of 1:1, measure the pH value and adjust it to 4.2 - 4.5, and filter and sterilize it with a 0.22 μm filter to obtain sodium citrate buffer. Note that it needs to be prepared and used immediately.

[0044] Weigh the STZ freeze-dried powder, place it in a sterile 50 mL centrifuge tube wrapped with tin foil paper, ice-bath, and add pre-cooled sodium citrate buffer (1% w / v) to dissolve it completely.

[0045] (2) Establishment of a T1DM rat model

[0046] Select male SD rats with a body weight of 200 ± 20 g, and perform modeling after 1 week of adaptive feeding. Fast for 10 h without water deprivation, and intraperitoneally inject STZ-sodium citrate buffer at a dosage of 65 mg / kg. The control group is intraperitoneally injected with sodium citrate buffer.

[0047] Three days after model establishment, tail tip blood was collected to measure random blood glucose for a total of 3 days. If the blood glucose was higher than 16.7 mmol / L for all 3 times and accompanied by obvious "polyphagia, polydipsia, polyuria and weight loss" symptoms, the model was considered successfully constructed. For those with unqualified blood glucose, STZ was supplemented at a dose of 10 - 20 mg / kg after the animal condition was stable, or after the rat blood glucose returned to normal, STZ was intraperitoneally injected again at the conventional dose (65 mg / Kg).

[0048] (3) Establish a model of ischemic foot ulcer in the lower limbs of T1DM rats

[0049] The established T1DM rats were continuously fed for 4 weeks to simulate the late stage of diabetes. Fast before surgery, anesthetize with 0.05 mg / kg of 1% amobarbital. Fix the rat supine and disinfect. Incise the skin along the course of the right inguinal blood vessels, carefully isolate the femoral vein and femoral artery, ligate their proximal and distal ends, cut and then suture the skin. After disinfection with iodophor, use a sterile biopsy punch to make a circular hole with a diameter of 0.7 mm on the rat's foot and take pictures to record the size of the ulcer wound surface.

[0050] Locally apply Cream (the control group applies excipient), with a thickness of 1 - 2 mm, once a day. Seal the wound surface with 3M breathable film and wrap it with a medical self-adhesive bandage to prevent it from falling off.

[0051] Take pictures to record the wound changes on the 0th, 3rd, 6th, 9th, 12th, 15th, 18th and 21st days respectively, and regularly observe to prevent the rat bandage from falling off. Calculate the wound healing situation through the wound healing rate. The wound healing rate = (A0 - Ax) / A0 × 100%, where A0 is the wound surface area on the 0th day and Ax is the wound surface area on the xth day.

[0052] Please refer to Figure 2 , compared with the model group, after locally applying Cream to the ulcer, the ulcer area was significantly reduced (P < 0.0001).

[0053] (4) Perfusion and tissue sampling

[0054] After anesthesia, the rat was fixed on the operating table. Cut open the abdominal cavity along the midline of the chest and abdomen, carefully cut through the diaphragm. After the lung retracted, expand the tear on both sides, then cut the ribs, clamp the xiphoid process with a hemostatic forceps and turn it outwards to fully open the chest cavity to expose the heart. After the perfusion needle was exhausted of air, insert it from the left ventricle, then cut open the right atrial appendage, and quickly perfuse with 0.9% normal saline until the color of the liver and mesenteric blood vessels became lighter or even white. If the tissue to be taken is used for tissue section, at this time, the normal saline should be replaced with 4% paraformaldehyde solution for continuous perfusion. It can be observed that the rat twitched and the rat tissue was fully fixed and hardened. Use a 10 mm biopsy punch to take tissue of a fixed size from the wound and store it in a -80°C refrigerator (the tissue for section should be immersed in 4% paraformaldehyde solution and stored in a 4°C refrigerator).

[0055] Example 2: Frozen section

[0056] (1) Fixation: The wound tissue was placed in 4% paraformaldehyde for 12 - 24 h for fixation.

[0057] (2) Sucrose gradient sedimentation of tissue: Prepare 10%, 20%, and 30% sucrose solutions in advance. The obtained tissue was soaked according to the gradient of increasing concentration. After the tissue settled to the bottom, it was replaced with the sucrose solution of the next concentration. After dehydration, it was stored in 30% sucrose solution in a 4°C refrigerator.

[0058] (3) OCT embedding: Pre-cool the cryostat in advance. The dehydrated tissue was placed on filter paper to absorb the moisture, then the tissue was put into a mold and OCT embedding medium was added, and it was placed on a quick-freezing rack for 30 min.

[0059] (4) Sectioning: The embedding stage with the wound tissue was fixed on the microtome. After trimming the frozen tissue, sections were made with a thickness of 8 μm, and the sections were adhered to the adhesive-free glass slides. Mark the glass slides and store them at -20°C for later use.

[0060] Example 3: Hematoxylin-eosin staining (HE staining)

[0061] (1) Take frozen sections of different groups. After rewarming at room temperature for 15 min, soak them in 4% paraformaldehyde solution for 10 min, and rinse with running water until the OCT is completely eluted.

[0062] (2) Stain with hematoxylin staining solution for 2.5 min and rinse with running water for 10 min.

[0063] (3) Differentiate with 1% hydrochloric acid ethanol for 2 - 30 s and rinse with running water for 10 min.

[0064] (4) Stain with eosin staining solution for 0.5 - 2 min.

[0065] (5) Dehydrate with 70% ethanol for 10 s, 80% ethanol for 10 s, 90% ethanol for 10 s, 100% ethanol for 10 s, soak in xylene I for 5 min, soak in xylene II for 5 min, and drop neutral gum for mounting.

[0066] (6) Air dry at room temperature, observe and photograph under the microscope.

[0067] Please refer to Figure 3 , compared with the model group, after local application of Cream on the ulcer, the re-epithelialization of the wound tissue increased significantly (P < 0.001).

[0068] Example 4: Masson staining

[0069] (1) The sections were fixed in 10% formalin solution for 1 h.

[0070] (2) Treat with Bouin's fixative overnight at room temperature and rinse with running water for 10 min.

[0071] (3) Prepare Weigert iron hematoxylin staining solution (A1:A2 = 1:1, freshly prepared before use), stain for 3 min, and rinse with running water for 30 s.

[0072] (4) Differentiate with acidic ethanol differentiating solution for 10 s and rinse with running water for 30 s.

[0073] (5) Blue with Masson bluing solution for 2 min and rinse with running water for 1 min.

[0074] (6) Stain with ponceau fuchsin staining solution for 2 min.

[0075] (7) Prepare weak acid working solution (distilled water:weak acid solution = 2:1), and wash with weak acid working solution for 1 min.

[0076] (8) Dropwise add phosphomolybdic acid solution and wash for 2 min, then wash with weak acid working solution for 1 min.

[0077] (9) Without pouring off the liquid on the slide, dropwise add aniline blue staining solution on the slide, stain for 2 min, and wash with weak acid working solution for 1 min.

[0078] (11) Dehydrate with 95% ethanol for 3 s, dehydrate with 100% ethanol 3 times, 10 s each time.

[0079] (12) Clear with xylene 3 times, 5 min each time, and mount with neutral balsam.

[0080] (13) Air dry at room temperature, observe and photograph under a microscope.

[0081] Please refer to Figure 4 , compared with the model group, after locally applying Cream to the ulcer, the content of collagen fibers in the wound tissue increased significantly (P < 0.001).

[0082] Example 5: Tissue RNA Extraction

[0083] (1) Take wound tissue or rat foot skin tissue of the same size, cut the tissue into small pieces as much as possible, add 1 mL of Trizol, and ultrasonically grind until there are no obvious tissue blocks in the EP tube, then incubate at room temperature for 10 min.

[0084] (2) Add 200 μL of chloroform, shake the EP tube vigorously for 15 s, incubate at room temperature for 3 min, centrifuge at 12000 rpm for 15 min at 4 °C.

[0085] (3) Slowly aspirate the upper clear and transparent aqueous phase into another new EP tube, add an equal volume of isopropanol, gently invert the EP tube up and down to mix well, and incubate overnight at -20 °C.

[0086] (4) Orient the EP tubes in the same direction and make marks, centrifuge at 12000 rpm × 15 min at 4 °C, and try to aspirate as much of the residual liquid in the EP tube as possible without touching the precipitate.

[0087] (5) Add 1 mL of pre-cooled 75% ethanol, resuspend the precipitate, then centrifuge at 7500 rpm × 5 min at 4 °C, and discard the supernatant.

[0088] (6) Repeat step (5) three times in total.

[0089] (7) Discard the supernatant, try to aspirate as much of the residual liquid in the tube as possible, and place it in the oven for 5 - 10 min until the precipitate becomes transparent and there are no obvious water droplets in the tube.

[0090] (8) Add 10 - 20 μL of DEPC water and place it in the oven for 5 - 10 min to promote RNA dissolution.

[0091] (9) Detect the RNA concentration with a UV spectrophotometer for subsequent experiments.

[0092] Example 6: Reverse transcription of tissue RNA

[0093] ① Use the Novoprotein reverse transcription kit and perform reverse transcription according to the instructions. After detecting the RNA concentration, add 1 μg of RNA to each well in an RNase-free eight-strip tube, and add RNase-free ddH2O to 16 μL, then immediately add 4 μL of DNA wiper, mix well and centrifuge.

[0094] ② Place the eight-strip tube in a PCR instrument set at 42 °C for 2 min.

[0095] ③ After completion, take out the eight-strip tube, add 4 μL of HiScript III RT SuperMix to each well, mix well and centrifuge.

[0096] ④ Place the eight-strip tube in a PCR instrument set at 37 °C for 15 min and 85 °C for 5 s. Obtain the final product cDNA, dilute it by an appropriate multiple for subsequent experiments.

[0097] Example 7: qPCR detection of the expression of pro-inflammatory factors IL-1β, IL-6 and TNF-α

[0098] Use the Novoprotein SYBR Green qPCR kit and perform qPCR experiments according to the instructions. The primers for IL-1β, IL-6 and TNF-α are as follows:

[0099] The reaction system is as follows:

[0100]

[0101] After the reaction, analyze the melting curve.

[0102] Table 2.1.2-5 Primers

[0103]

[0104] Please refer to Figure 5 , compared with the model group, after locally applying Cream to the ulcer, the expressions of pro-inflammatory factors IL-1β, IL-6 and TNF-α were significantly decreased (P < 0.01).

[0105] In summary, in this application, a rat lower limb ischemic type 1 DFU model was constructed, and the applicability of the Tangzukang ointment provided in this application to various DFU dialectical types was verified through the above model; and through dosage form improvement, it can be directly administered to the wound surface, increasing the curative effect, reducing the irritation to the wound surface, and enhancing the compliance of patients. Cream is a multi-component traditional Chinese medicine: it can improve the anti-inflammatory effect; promote the regeneration of wound tissue; and accelerate the healing of the ulcer surface.

Claims

1. A Tangzukang cream for promoting the healing of diabetic foot ulcer wounds, characterized in that: The cream contains the following traditional Chinese medicine components: Atractylodes lancea, Phellodendron amurense, Coix lacryma-jobi, Cinnamomum cassia, Angelica sinensis, Astragalus membranaceus, and Borneol.

2. The sugar foot health cream according to claim 1, characterized in that: The mass ratio of the traditional Chinese medicine components is as follows: Atractylodes lancea 200 parts, Phellodendron amurense 200 parts, Coix lacryma-jobi 600 parts, Cinnamomum cassia 60 parts, Angelica sinensis 300 parts, Astragalus membranaceus 300 parts, and Borneol 4 parts.

3. The sugar foot health cream according to claim 1, characterized in that: The cream also includes materials for preparing the oil phase: Stearic acid, Glycerol monostearate, Light liquid paraffin, Ethylparaben, Polysorbate 80, Sorbitan oleate, and Benzoic acid.

4. The sugar foot health cream according to claim 1, characterized in that: The cream also includes glycerol and purified water.

5. The Tangzukang Cream according to claim 1, wherein: The preparation process of the cream is as follows: S1: Weigh the formula amount of Atractylodes lancea, Phellodendron amurense, Coix lacryma-jobi, Cinnamomum cassia, Angelica sinensis, and Astragalus membranaceus. Add 8 times the amount of water for soaking, decoct, and filter; add 6 times the amount of water to the filter residue for decocting and filtering, and combine the two filtrates; concentrate the filtrate into an extract for standby, dry, pulverize, and reserve. S2: Prepare the oil phase. S3: Place the extract in S1 and purified water in a container for heating. While stirring, add glycerol, and let it stand to obtain the water phase. S4: Add the water phase in S3 to the oil phase in S2 and mix evenly to obtain the Tangzukang Ointment.

6. The Tangzukang Cream according to claim 1, wherein: The method for preparing the oil phase in S2 is as follows: Weigh stearic acid, glycerol monostearate, light liquid paraffin, ethylparaben, polysorbate 80, sorbitan oleate, benzoic acid, and borneol. After weighing, place the above materials in a dry container, stir, and heat to 78°C - 82°C until completely dissolved, then let it stand for standby.

7. The sugar foot health cream according to claim 1, characterized in that: The heating temperature in S3 is 78°C - 82°C.

8. Use of the Tangzukang cream according to any one of claims 1 - 7 in the preparation of a drug for promoting the healing of diabetic foot ulcer wounds.

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