Traditional Chinese medicine composition for treating burns and scalds

Abstract

The application provides a traditional Chinese medicine composition for treating burns and scalds, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition for treating burns and scalds is prepared from the roots of Euphorbia humifusa and Vitis thunbergii according to certain matching, and has the following effects: (1) pain relief: pain is relieved or even disappears within 15 minutes; (2) convergence: the exudation of tissue fluid can be stopped within two hours; (3) bacteriostasis: the wounds of burns and scalds or ulceration for months can gradually heal after use, and the necrotic tissue will scab and fall off; (4) muscle regeneration: the patients with burns and scalds do not need skin grafting, and scar tissue proliferation is avoided, so that the disability rate of the patients is greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition for treating burns and scalds. Background Technology

[0002] Burns are injuries to the skin or other organs and tissues caused primarily by high temperatures, or by radiation, electricity, friction, or contact with chemicals. Thermal (high temperature) burns occur when some or all of the cells in the skin or other tissues are destroyed by hot liquids (scalds), hot solids (contact burns), or flames (burns).

[0003] Based on the degree of injury, burns can be classified into superficial (first-degree burns), superficial partial-layer burns (superficial second-degree burns), deep partial-layer burns (deep second-degree burns), and full-layer burns (third-degree burns). For these non-life-threatening burns, the wound is usually rinsed with copious amounts of sterile isotonic saline. If necessary, 0.1-0.2% povidone-iodine (iodophor) or 1:2000 chlorhexidine can be applied for cleaning and application. Cold therapy can be administered simultaneously with debridement (suitable for mild to moderate burns). After debridement, exposure or bandaging may be used as appropriate. Deep burns generally refer to deep second-degree, third-degree, or higher burns. In third-degree burns, necrotic tissue is called eschar, and in second-degree burns, necrotic tissue is called eschar. Treatment typically involves escharotomy and skin grafting, eschar removal and skin grafting, eschar removal, and skin grafting.

[0004] The aforementioned treatments are often accompanied by severe pain; patients with deep burns may experience lifelong pain even after skin grafting. Burns cause significant tissue fluid exudation, and patients with large-area burns may even experience shock, endangering their lives. Burn wounds are prone to recurrent infections and suppuration, and even after healing, scar hyperplasia can lead to disfigurement and disability in severe cases. Topical medications can reduce these problems, thus playing an important role in burn treatment. Studies have shown that traditional Chinese medicine has significant effects in treating burns.

[0005] For example, patent CN103479917A discloses a traditional Chinese medicine composition for treating burns, which is made from bamboo shoots, dragon's blood, purple gromwell root, four-sided hemp, large thistle, arborvitae leaves, artemisia annua, lindera leaves, vine, coptis, snake slough, lead oxide, and sesame oil in a specific ratio. Another example is patent CN102846778A, which discloses a traditional Chinese medicine for treating burns and its preparation method, which is made from snakeberry, lobelia chinensis, and rosehip leaves in a specific ratio. After washing, drying, and pulverizing, it is mixed with an appropriate amount of sesame oil to obtain the medicine. It has anti-inflammatory, analgesic, heat-clearing, detoxifying, astringent, antibacterial, and tissue-regenerating effects, and has a certain ability to penetrate scabs and eschars, effectively treating burns without leaving scars. For example, patent CN115444811A discloses a topical borneol camphor oil preparation for treating burns and its preparation method. It is prepared by combining borneol camphor oil, glycerin, carbomer 940, chitosan, 1,3-butanediol, preservatives, neutralizing agents, solubilizers, penetration enhancers, ethanol, and deionized water in a specific formulation. The use of borneol camphor oil as the main ingredient can improve the healing effect and shorten the treatment time. However, existing traditional Chinese medicine formulas have limited efficacy in clinical applications, and the problems of pain, ulceration, and scar hyperplasia after burns are difficult to resolve. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a traditional Chinese medicine composition for treating burns and scalds, which is made from the root of *Rhizoma Cypripedii* and the root of *Rhizoma Cypripedii* in a specific combination. This composition has analgesic, astringent, antibacterial, and tissue-regenerating effects, and is highly stable, effectively treating burns and scalds without leaving scars.

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

[0008] A traditional Chinese medicine composition for treating burns and scalds, comprising fresh *Rhizoma Cyprinus serratus* root and *Rhizoma Cyprinus coccinea* root.

[0009] A traditional Chinese medicine composition for treating burns and scalds, comprising fresh thorn root, grape root, and camphor water or natural borneol.

[0010] Furthermore, the traditional Chinese medicine composition for treating burns and scalds contains, by weight percentage, the following raw materials: 40-90% of *Rhizoma Cyathulae* root, 9-59% of *Rhizoma Cyathulae* root, and 1-15% of *Cinnamomum camphora* water.

[0011] Preferably, the traditional Chinese medicine composition for treating burns comprises the following ingredients by weight percentage: 70% *Rhizoma Cyathulae* root, 20% *Rhizoma Cyathulae* root, and 10% *Cinnamomum camphora* water.

[0012] Preferably, the traditional Chinese medicine composition for treating burns comprises the following ingredients by weight percentage: 40% *Rhizoma Cyathulae* root, 59% *Rhizoma Cyathulae* root, and 1% *Cinnamomum camphora* water.

[0013] Preferably, the traditional Chinese medicine composition for treating burns contains the following ingredients by weight percentage: 85% *Rhizoma Cyathulae* root, 10% *Rhizoma Cyathulae* root, and 5% *Cinnamomum camphora* water.

[0014] Preferably, the traditional Chinese medicine composition for treating burns and scalds contains the following raw materials by weight percentage: 40-90% of *Rhizophora stylosa* root, 10-60% of *Vitis thunbergii* root, and 1‰-5‰ of natural borneol, which accounts for 1‰-5‰ of the total amount of *Rhizophora stylosa* root and *Vitis thunbergii* root.

[0015] More preferably, the traditional Chinese medicine composition for treating burns contains the following raw materials by weight percentage: 77.8% of *Rhizoma Cyprinus serratus* root, 22.2% of *Rhizoma Cyprinus rubra* root, and 2.5‰ of natural borneol, which accounts for 2.5‰ of the total amount of *Rhizoma Cyprinus serratus* root and *Rhizoma Cyprinus rubra* root.

[0016] The root of *Cirsium chlorolepis* Petrak ex Hand.-Mazz, a plant in the genus *Cirsium* of the Asteraceae family, can be harvested year-round, but fresh roots harvested in autumn are considered the most effective. After harvesting, the fresh roots should be washed, drained, and refrigerated at -20°C for later use. *Cirsium chlorolepis* root has cooling, analgesic, antipruritic, antibacterial, tissue-regenerating, and pus-draining properties.

[0017] Jade grape root is the root of *Ampelopsis delavayana* Planch, a plant belonging to the genus *Ampelopsis* in the family Vitaceae. Fresh roots harvested in autumn are considered the most effective. After harvesting, wash, drain, and remove the core from the fresh roots, then refrigerate at -20°C for later use. Jade grape root has analgesic, antiseptic, anti-inflammatory, detoxifying, and anti-inflammatory properties.

[0018] In the traditional Chinese medicine composition for treating burns and scalds of the present invention, the camphor water is prepared by the following method: the branches and leaves of camphor tree are dried, pulverized and then distilled. The oil-water mixture remaining after distillation and the aqueous phase after oil-water separation are the camphor water.

[0019] Furthermore, the content of dextrorotatory borneol in the camphor water is 0.1-0.5 mg / mL.

[0020] The preparation method of the above-mentioned traditional Chinese medicine composition for treating burns includes the following steps:

[0021] (1) Fresh purslane root and grape root were added to water, crushed and then separated into liquid I and solid. The solid was dried and crushed into powder of 800-1500 mesh. Liquid I was separated into liquid II and slurry. Finally, the powder and slurry were mixed to obtain a mixture.

[0022] (2) Add camphor water or natural borneol to the mixture in step (1), mix thoroughly to obtain the original drug intermediate, and refrigerate for later use.

[0023] Further, in step (1), the step of pulverizing is as follows: fresh spur roots or grape roots are added to water and pulverized to 80-100 mesh, and the solid and liquid are separated to obtain liquid I and solid matter; after the solid matter is dried, it is pulverized step by step to 200 mesh and 800 mesh, and finally pulverized to 800-1500 mesh powder; liquid I is separated to obtain liquid II and slurry, and the volume of liquid II is 60%-80% of the volume of liquid I; finally, the powder and slurry are mixed to obtain a mixture.

[0024] Furthermore, in step (1), the amount of water added is 2-10 times the mass of fresh spur roots or grape roots.

[0025] Furthermore, in step (2), the resulting refrigeration temperature is 4-8°C, preferably 4°C.

[0026] The above-mentioned traditional Chinese medicine composition for treating burns and scalds is used in the preparation of medicines for treating burn and scald diseases, which include wound infection, fluid exudation, edema and pain symptoms caused by burns and scalds.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) This invention uses fresh thorn root and grape root in a certain combination to prepare a traditional Chinese medicine composition for treating burns and scalds. Through the synergistic effect of fresh thorn root and grape root, the traditional Chinese medicine composition has the following effects: analgesia: pain is relieved or even disappears within 15 minutes, and analgesics can be discontinued; astringency: it can stop the exudation of tissue fluid within 2 hours; antibacterial: after use, the wound can gradually heal even for fresh burns or wounds that have been ulcerated for several months, and necrotic tissue will scab and fall off; tissue regeneration: burn patients do not need skin grafts, and there is no hyperplasia of scar tissue, which greatly reduces the disability rate of patients.

[0029] (2) The traditional Chinese medicine composition of the present invention also has good stability.

[0030] (3) The camphor water of the present invention is an aqueous solution remaining after the branches and leaves of camphor tree are extracted and processed into natural borneol. The addition of camphor water can improve the healing effect of the affected area, shorten the treatment time, and improve stability. Attached Figure Description

[0031] Figure 1 The effect of the antibacterial cream on HaCaT cells was observed under a light microscope;

[0032] Figure 2 The effect of the antibacterial solution on HaCaT cells was observed under a light microscope.

[0033] Figure 3 It is the effect of the antibacterial cream on the migration of HaCaT cells;

[0034] Figure 4 It is the effect of the antibacterial solution on the migration of HaCaT cells;

[0035] Figure 5 The effect of the antibacterial solution on HaCaT cell apoptosis was observed using TUNEL staining.

[0036] Figure 6 The effect of the antibacterial solution on the proliferation of HaCaT cells was observed using the CCK8 assay.

[0037] Figure 7 The effect of the antibacterial cream on HaCaT cell senescence was observed using β-gal staining.

[0038] Figure 8 The effect of the antibacterial solution on the senescence of HaCaT cells was observed using β-gal staining.

[0039] Figure 9 The effect of the antibacterial cream on HFF cells was observed under a light microscope;

[0040] Figure 10 The effect of the antibacterial solution on HFF cells was observed under a light microscope;

[0041] Figure 11 It is the effect of the antibacterial cream on HFF cell migration;

[0042] Figure 12 It is the effect of the antibacterial solution on the migration of HFF cells;

[0043] Figure 13 The effect of the antibacterial solution on HFF cell apoptosis was detected by flow cytometry.

[0044] Figure 14 The effect of the antibacterial cream on HFF cell apoptosis was observed using TUNEL staining.

[0045] Figure 15 The effect of the antibacterial solution on HFF cell apoptosis was observed using TUNEL staining.

[0046] Figure 16 The effect of the antibacterial solution on HFF cell proliferation was observed using the CCK8 assay.

[0047] Figure 17 The effect of the antibacterial cream on HFF cell senescence was observed using β-gal staining.

[0048] Figure 18 The effect of the antibacterial solution on HFF cell senescence was observed using β-gal staining.

[0049] Figure 19 The effect of the antibacterial cream on HUVEC cells was observed under a light microscope;

[0050] Figure 20The effect of the antibacterial solution on HUVEC cells was observed under a light microscope;

[0051] Figure 21 This refers to the effect of the antibacterial cream on the migration of HUVEC cells;

[0052] Figure 22 It is the effect of the antibacterial solution on the migration of HUVEC cells;

[0053] Figure 23 The effect of the antibacterial solution on HUVEC cell apoptosis was detected by flow cytometry.

[0054] Figure 24 The effect of the antibacterial solution on the proliferation of HUVEC cells was observed using the CCK8 assay.

[0055] Figure 25 The effect of the antibacterial cream on HUVEC cell senescence was observed using β-gal staining.

[0056] Figure 26 The effect of the antibacterial solution on HUVEC cell senescence was observed using β-gal staining.

[0057] Figure 27 This is the establishment of a rat model of deep second-degree burns;

[0058] Figure 28 The effect of antibacterial cream on the mechanical pain threshold of rats with deep second-degree burns was investigated, with N=6, **, P<0.01 compared with the normal group; ##, P<0.01 compared with the model group;

[0059] Figure 29 The effect of antibacterial cream on the thermal pain threshold of rats with deep second-degree burns was investigated, with N=4, **, P<0.01 compared with the normal group; ##, P<0.01 compared with the model group;

[0060] Figure 30 These are photos of the wounds of rats in each group 7 days after administration. 1 is the model group, 2 is the positive drug group, 3 is the low-dose antibacterial solution group, 4 is the high-dose antibacterial solution group, 5 is the low-dose antibacterial cream group, 6 is the high-dose antibacterial cream group, 7 is the low-dose powder group, and 8 is the high-dose powder group.

[0061] Figure 31 The effect of antibacterial liquid, antibacterial cream and powder on the burn area of ​​deep second-degree burns in rats (administered for 7 days)**, P<0.01, *, P<0.05 compared with the model group;

[0062] Figure 32 HE staining was used to observe the pathological changes in rat skin wound tissue (3 days after drug administration), where A. 4×, B. 10×;

[0063] Figure 33The purpose of this study was to observe the pathological changes in rat skin wound tissues using HE staining (after 7 days of drug administration), where A. 4× and B. 10× were observed.

[0064] Figure 34 Masson staining was used to observe the pathological changes in rat skin wound tissue (3 days after drug administration). A. 4×, B. 10×; Yellow arrow: damaged sebaceous glands and hair follicles, red arrow: inflammatory cell exudation;

[0065] Figure 35 Masson staining was used to observe the pathological changes in rat skin wound tissue (7 days after drug administration). A. 4×, B. 10×; Yellow arrow: damaged sebaceous glands and hair follicles, black arrow: collagen deposition, red arrow: inflammatory cell exudation, green arrow: separation of epidermis and dermis.

[0066] Figure 36 This is an injury diagram from a typical case 1 of the present invention;

[0067] Figure 37 This is an injury diagram from a typical case 2 of the present invention;

[0068] Figure 38 This is an injury diagram from a typical case 3 of the present invention;

[0069] Figure 39 This is an injury diagram of typical case 4 of the present invention;

[0070] Figure 40 This is the injury diagram of typical case 5 of the present invention. Detailed Implementation

[0071] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following description is merely an exemplary illustration of the scope of protection of the present invention, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection of the present invention.

[0072] Unless otherwise specified, all chemical reagents used in the embodiments of this invention were obtained through conventional commercial means.

[0073] Example 1

[0074] A traditional Chinese medicine composition for treating burns and scalds is prepared by the following method: 700g of fresh *Rhizoma Cypripedii* root and 200g of *Rhizoma Cypripedii* root are mixed with 4g of water. The mixture is first pulverized to 100 mesh, and then separated into a solid and a liquid I. The particulate matter in liquid I is larger than 800 mesh. After drying, the solid is pulverized in stages at 200 mesh and 800 mesh, and finally pulverized into a powder of 1000 mesh. Liquid I is further separated into a liquid II and a slurry, with the volume of liquid II being 70% of the volume of liquid I. The powder and the slurry are mixed, and 100g of *Cinnamomum camphora* water is added. After thorough mixing, the intermediate of the original medicine is obtained and refrigerated at 4°C for later use.

[0075] The method for making camphor water is as follows: the branches and leaves of camphor tree are dried, crushed and then distilled. The oil and water mixture remaining after distillation is the aqueous phase after oil and water separation, which is the camphor water.

[0076] Example 2

[0077] A traditional Chinese medicine composition for treating burns and scalds is prepared by the following method: 400g of fresh *Rhizoma Cypripedii* root and 590g of *Rhizoma Cypripedii* root are mixed with 4g of water. The mixture is first pulverized to 100 mesh, and then separated into a solid and a liquid I. The particulate matter in liquid I is larger than 800 mesh. After drying, the solid is pulverized in stages at 200 mesh and 800 mesh, and finally pulverized into a powder of 1000 mesh. Liquid I is then separated into a liquid II and a slurry, with the volume of liquid II being 68% of the volume of liquid I. The powder and slurry are mixed, and 10g of camphor tree water is added. The camphor tree water is prepared in the same way as in Example 1. After thorough mixing, the intermediate of the original medicine is obtained and refrigerated at 4°C for later use.

[0078] Example 3

[0079] A traditional Chinese medicine composition for treating burns and scalds is prepared by the following method: 850g of fresh *Rhizoma Cypripedii* root and 100g of *Rhizoma Cypripedii* root are mixed with 4g of water. The mixture is first pulverized to 100 mesh, and the solid and liquid are separated to obtain a solid and liquid I. The particulate matter in liquid I has a mesh size greater than 800 mesh. After drying, the solid is pulverized in stages at 200 mesh and 800 mesh, and finally pulverized to 1000 mesh powder. Liquid I is separated to obtain liquid II and a slurry. The volume of liquid II is 76% of the volume of liquid I. The powder and slurry are mixed, and 50g of camphor tree water is added. The method for preparing camphor tree water is the same as in Example 1. After thorough mixing, the intermediate of the original medicine is obtained and refrigerated at 4°C for later use.

[0080] Example 4

[0081] The difference from Example 1 is that the camphor water is replaced with natural borneol, otherwise it is the same as Example 1. Specifically, a traditional Chinese medicine composition for treating burns is prepared by the following method: 700g of fresh horse thorn root and 200g of grape root are mixed with 4g of water, pulverized to 100 mesh, and separated into solid and liquid components to obtain solid and liquid I, wherein the particles in liquid I are larger than 800 mesh. After drying, the solid is pulverized in stages of 200 mesh and 800 mesh, and finally pulverized into powder of 1000 mesh. Liquid I is further separated into liquid II and slurry, the volume of liquid II being 70% of the volume of liquid I. The powder and slurry are mixed, and 2.5‰ of the weight of natural borneol is added to the mixture. After thorough mixing, the intermediate of the original medicine is obtained and refrigerated at 4°C for later use.

[0082] Example 5

[0083] Preparation of the antibacterial solution. The antibacterial solution is prepared using conventional techniques, namely, by adding purified water and sodium benzoate to the drug intermediate from Example 1.

[0084] Example 6

[0085] Preparation of antibacterial cream. The preparation method of antibacterial cream is also a conventional technique. Using the original drug intermediate of Example 1, carbomer and sodium benzoate are added to obtain the antibacterial cream.

[0086] Example 7

[0087] Preparation of antibacterial powder. The preparation method of antibacterial powder is also a conventional technique. The active pharmaceutical ingredient intermediate of Example 1 is dried and then pulverized to obtain antibacterial powder.

[0088] Comparative Examples 1-4

[0089] The difference between Comparative Examples 1-4 and Example 1 is that the roots of *Vitis vinifera* were replaced with *Schefflera heptaphylla*, *Ampelopsis japonica*, *Rosa laevigata*, and *Rubia cordifolia*, respectively, in the comparative examples; otherwise, they were the same as in Example 1. The intermediates prepared in Example 1 and the comparative examples were used to treat burns. At least 50 people in each group had used the drugs. The efficacy was scored based on four dimensions: analgesia, astringency, antibacterial effect, and tissue regeneration. Example 1 was scored out of 5, and higher scores in the other examples indicated better efficacy. The results are shown in Table 1.

[0090] Table 1.

[0091]

[0092]

[0093] Experimental Example 1

[0094] Effects of the antibacterial solution of Example 5 and the antibacterial cream of Example 6 on epidermal cells, endothelial cells and fibroblasts

[0095] Epidermal cells (HaCaT cells), endothelial cells (HUVEC cells), and fibroblasts (HFF cells) were cultured in vitro and treated with different doses of antibacterial solution (10 μL / mL, 50 μL / mL, 100 μL / mL) and antibacterial cream (0.01 mg / mL, 0.05 mg / mL, 0.10 mg / mL). The effects of antibacterial solution and antibacterial cream on cell morphology were observed under a light microscope. The effects of antibacterial solution and antibacterial cream on cell migration ability were observed through cell scratch assay. The effects of antibacterial solution and antibacterial cream on cell apoptosis were observed through flow cytometry and TUNEL staining. The effects of antibacterial solution and antibacterial cream on cell proliferation were detected by CCK8 assay. The effects of antibacterial solution and antibacterial cream on cell senescence were observed by β-gal staining.

[0096] The cells included the following three types: epidermal cells: human immortalized keratinocytes (HaCaT cells), purchased from the Cell Bank of the Chinese Academy of Sciences (catalog number: SCSP-5091); endothelial cells: human umbilical vein endothelial cells (HUVEC cells), purchased from the American Type Culture Collection (ATCC); and fibroblasts: human skin fibroblasts (HFF cells), purchased from the Cell Bank of the Chinese Academy of Sciences (catalog number: SCSP-106).

[0097] Cell culture

[0098] Cell thawing: Remove the cryopreserved cell tubes and place them in a 37°C water bath, gently shaking to rapidly thaw them. Then, thoroughly disinfect the outside of the tubes with alcohol before transferring them to a biosafety cabinet. Transfer the cell suspension to a 15mL centrifuge tube, slowly adding complete cell culture medium until the suspension reaches 5-6mL. Seal the tube tightly and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, retaining the cell pellet. Add an appropriate amount of complete culture medium to the centrifuge tube, gently pipetting the cell pellet 7-8 times to prepare a homogeneous cell suspension. Transfer the suspension to a culture dish and incubate in a cell culture incubator. Observe cell adhesion and growth regularly.

[0099] Cell passage: Once cells reach a confluence greater than 70%-80%, passage them immediately. After discarding the supernatant, rinse the cells with an appropriate amount of PBS to remove dead cells and residual serum. Then, add trypsin for approximately 30 seconds, followed by the addition of serum to stop the digestion. Gently pipette the cells into a homogeneous single-cell suspension, transfer to a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, retaining the cell pellet. Add an appropriate amount of complete culture medium to prepare a cell suspension. Add the suspension to new cell culture dishes according to experimental requirements, place in a cell culture incubator, and observe cell adhesion and growth regularly to complete subsequent experiments.

[0100] Cell flow cytometry analysis to detect cell apoptosis

[0101] Cells were seeded in six-well plates, and the drug was added for culture. The culture medium was aspirated from each well, and pre-chilled PBS was added. The plates were gently shaken to wash away floating cells. Accutase enzyme solution was added, and the plates were incubated for 1-2 minutes for digestion. Once the cells had completely floated, serum was added to neutralize and terminate the digestion. The cell suspension was collected in centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The cell pellet was then washed 2-3 times with PBS. The cells were resuspended in 1x Binding Buffer diluted to 500 μL, followed by 5 μL of Annexin V-APC and 10 μL of 7-AAD. The mixture was gently vortexed and incubated at room temperature in the dark for 5 minutes. Subsequently, Annexin V-APC was detected using the APC detection channel and 7-AAD was detected using the PI detection channel on a flow cytometer.

[0102] Cell scratch assay

[0103] First, prepare a sterile 6-well plate. Using a marker pen, evenly streak three lines on the bottom of each well, spaced 1 cm apart. Then, seed cells into the 6-well plate and add the test drug. When the cell confluence reaches 90% or higher, use a 200 μL pipette tip perpendicular to the bottom of the well and evenly and firmly streak the cells along the marker pen lines. Rinse off any detached cells with PBS. Add 2 mL of culture medium containing 0.5% FBS to each well. Observe and photograph the cell position around the streaks under a microscope, recording this as the 0h cell position. Continue culturing the 6-well plate, and photograph the cell position again after 24 hours.

[0104] CCK8 method for detecting cell proliferation

[0105] Cells were seeded in 96-well plates at a density of 5000 cells per well, and the plates were incubated in a cell culture incubator for 4–6 hours. After cell attachment, 10 μL of CCK-8 solution was added to each well, and the plates were incubated for another 2 hours. The absorbance at 450 nm was measured using a microplate reader and recorded; this was the data at 0 h. Absorbance was subsequently measured at 6 h, 12 h, and 24 h of incubation.

[0106] β-gal staining (galactosidase staining)

[0107] Aspirate the cell culture medium, wash the cells once with PBS, add 1 mL of β-galactosidase staining and fixing solution, and fix at room temperature for 15 minutes. Aspirate the cell fixative, wash the cells three times with PBS for 3 minutes each time. Aspirate the PBS, add 1 mL of staining working solution to each well (preparation method shown in Table 2), seal the 6-well plate with parafilm, and incubate overnight at 37°C. Observe under a regular optical microscope.

[0108] Table 2. Preparation method of staining working solution

[0109] β-galactosidase staining solution B 10μL β-galactosidase staining solution C 930μL X-Gal solution 50μL total 1000μL

[0110] TUNEL staining method for detecting cell apoptosis

[0111] Cells were washed once with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed once with PBS, and incubated with PBS containing 0.3% Triton X-100 at room temperature for 5 minutes. Cells were washed twice with PBS, and 50 μL of TUNEL detection solution (preparation method shown in Table 3) was added to the sample. The sample was incubated at 37°C in the dark for 60 minutes, washed three times with PBS, mounted with anti-fluorescence quenching mounting solution, and observed under a fluorescence microscope.

[0112] Table 3. Preparation method of TUNEL test solution

[0113] Fluorescent labeling solution 45μL TUNEL test solution 50μL

[0114] (1) Effects of antibacterial liquids and creams on epidermal cells

[0115] a. Observing the effects of antibacterial solutions and creams on epidermal cells under a light microscope

[0116] After adding different concentrations of antibacterial cream to HaCaT cells cultured in vitro, some cells underwent transformation, exhibiting a fibroblast-like structure. Figure 1 (As shown by the red arrow), suggesting that the epidermal cells may have undergone a transformation into epithelial-mesenchymal cells.

[0117] After adding different concentrations of antibacterial solution to HaCaT cells cultured in vitro, some cells also underwent transformation. Figure 2 (As shown by the red arrow), suggesting that the epidermal cells may have undergone a transformation into epithelial-mesenchymal cells.

[0118] b. The effect of antibacterial creams and solutions on epidermal cell migration

[0119] The effect of antibacterial cream on epidermal cell migration was observed through a scratch test. Figure 3 As shown, after 24 hours of antibacterial cream application, the intercellular spaces of the 0.05 mg / mL antibacterial cream were smaller, suggesting that the antibacterial cream promotes the migration of epidermal cells.

[0120] The effect of the antibacterial solution on epidermal cell migration was observed through a scratch test. Figure 4 As shown, after 24 hours of treatment with the antibacterial solution, the intercellular spaces were smaller in the 50 μL / mL and 100 μL / mL antibacterial solution groups, suggesting that the antibacterial solution promotes the migration of epidermal cells.

[0121] c. Effects of antibacterial creams and solutions on epidermal cell apoptosis

[0122] The effect of the antibacterial solution on epidermal cell apoptosis was observed by TUNEL staining, such as Figure 5 As shown, there was no significant change in the apoptosis rate among the groups.

[0123] d. Effects of antibacterial solution on epidermal cell proliferation

[0124] The effect of antibacterial solutions on epidermal cell proliferation was detected using the CCK8 assay. The results showed that different concentrations of antibacterial solutions all promoted epidermal cell proliferation. Figure 6 ).

[0125] e. The effects of antibacterial solutions and creams on epidermal cell aging

[0126] The effect of the antibacterial cream on epidermal cell senescence was observed using β-gal staining. No increase in stained epidermal cells was found, suggesting that the antibacterial cream may not affect epidermal cell senescence. Figure 7 ).

[0127] The effect of the antibacterial solution on epidermal cell senescence was observed using the β-gal staining method. No increase in stained epidermal cells was found, suggesting that the antibacterial solution may not affect epidermal cell senescence. Figure 8 ).

[0128] (2) Effects of antibacterial solutions and antibacterial creams on fibroblasts

[0129] a. Observation of the effects of antibacterial solutions and creams on fibroblasts under a light microscope

[0130] No significant changes were observed in HFF cells cultured in vitro after the addition of the antibacterial cream. Figure 9 ).

[0131] No significant changes were observed in HFF cells cultured in vitro after the addition of the antibacterial solution. Figure 10 ).

[0132] b. Effects of antibacterial creams and solutions on fibroblast migration

[0133] The effect of the antibacterial cream on fibroblast migration was observed through a scratch assay. Figure 11 As shown, after 24 hours of antibacterial cream application, the intercellular spaces in the 0.05 mg / mL antibacterial cream group were smaller, suggesting that the antibacterial cream promotes fibroblast migration.

[0134] The effect of the antibacterial solution on fibroblast migration was observed through a scratch assay. Figure 12 As shown, after 24 hours of treatment with the antibacterial solution, the intercellular spaces were smaller in the 50 μL / mL and 100 μL / mL antibacterial solution groups, suggesting that the antibacterial solution promotes the migration of fibroblasts.

[0135] c. Effects of antibacterial solutions and creams on fibroblast apoptosis

[0136] The effect of antibacterial solution on fibroblast apoptosis was observed by flow cytometry, such as Figure 13 As shown, there were no significant changes in the apoptosis rate of cells in each group, suggesting that the antibacterial solution may not have any effect on fibroblast apoptosis.

[0137] The effect of the antibacterial cream on fibroblast apoptosis was observed by TUNEL staining, such as Figure 14 As shown, there were no significant changes in the apoptosis rate of cells in each group, suggesting that the antibacterial cream may not have any effect on fibroblast apoptosis.

[0138] The effect of the antibacterial solution on fibroblast apoptosis was observed by TUNEL staining, such as Figure 15 As shown, there were no significant changes in the apoptosis rate of cells in each group, suggesting that the antibacterial solution may not have any effect on fibroblast apoptosis.

[0139] c. Effects of antibacterial solution on fibroblast proliferation

[0140] The effect of antibacterial solutions on fibroblast proliferation was detected using the CCK8 assay. The results showed that different concentrations of antibacterial solutions promoted fibroblast proliferation. Figure 16 ).

[0141] d. Effects of antibacterial solutions and creams on fibroblast senescence

[0142] The effect of the antibacterial cream on fibroblast senescence was observed using β-gal staining. No increase in stained fibroblasts was found, suggesting that the antibacterial cream may not affect fibroblast senescence. Figure 17 ).

[0143] The effect of the antibacterial solution on fibroblast senescence was observed using β-gal staining. No increase in stained fibroblasts was found, suggesting that the antibacterial solution may not affect fibroblast senescence. Figure 18 ).

[0144] (3) Effects of antibacterial solutions and antibacterial creams on endothelial cells

[0145] a. Observation of the effects of antibacterial solutions and creams on endothelial cells under a light microscope

[0146] When an antibacterial cream was added to HUVEC cells cultured in vitro, the number of HUVEC cells decreased after treatment with high concentrations of the antibacterial cream. Figure 19 ).

[0147] When an antibacterial solution was added to HUVEC cells cultured in vitro, the number of HUVEC cells decreased after treatment with a high concentration of the antibacterial solution. Figure 20 ).

[0148] b. Effects of antibacterial creams and solutions on endothelial cell migration

[0149] The effect of the antibacterial cream on HUVEC cell migration was observed through a scratch assay. Figure 21 As shown, after 24 hours of antibacterial cream application, the intercellular spaces in the 0.10 mg / mL antibacterial cream group were smaller, suggesting that the antibacterial cream promotes the migration of HUVEC cells.

[0150] The effect of the antibacterial solution on endothelial cell migration was observed through a scratch test, such as... Figure 22 As shown, after 24 hours of treatment with the antibacterial solution, the differences in intercellular spaces among the groups were small, suggesting that the antibacterial solution had a weak effect on the migration of endothelial cells.

[0151] c. Effects of antibacterial solutions and creams on endothelial cell apoptosis

[0152] The effect of the antibacterial solution on endothelial cell apoptosis was observed by flow cytometry, such as Figure 23 As shown, there was no significant change in the apoptosis rate among the groups.

[0153] d. Effects of antibacterial solution on endothelial cell proliferation

[0154] The effect of antibacterial solutions on endothelial cell proliferation was detected using the CCK8 assay. The results showed that different concentrations of antibacterial solutions promoted endothelial cell proliferation. Figure 24 ).

[0155] e. The effects of antibacterial solutions and creams on endothelial cell senescence

[0156] The effect of the antibacterial cream on endothelial cell senescence was observed using β-gal staining. No increase in stained endothelial cells was found, suggesting that the antibacterial cream may not affect endothelial cell senescence. Figure 25 ).

[0157] The effect of the antibacterial solution on endothelial cell senescence was observed using β-gal staining. No increase in stained endothelial cells was found, suggesting that the antibacterial solution may not affect endothelial cell senescence. Figure 26 ).

[0158] Experiments show that antibacterial solutions and creams can promote epidermal cell transformation and migration, which helps to promote the migration of new skin grafts during burn recovery; antibacterial solutions and creams promote fibroblast proliferation, which helps to promote the formation of granulation tissue in burn wounds.

[0159] Experimental Example 2

[0160] The therapeutic effects of the antibacterial liquid of Example 5, the antibacterial cream of Example 6, and the antibacterial powder of Example 7 on scalded rats.

[0161] Positive control drug information: Silver sulfadiazine cream, 500g: 5g, manufacturer: Guangdong Hengjian Pharmaceutical Co., Ltd.

[0162] Experimental animals and experimental models: Male SD rats (200-220g) were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. After purchase, the SD rats were acclimatized for one week, then randomly divided into groups. Hair removal was performed by applying 5% sodium sulfide to the rats' backs. The rats were then anesthetized with a small animal anesthetic, and a 2.5cm diameter metal object was brought into contact with the rats for 15 seconds, creating a deep II-shaped wound (circular or oval) approximately 2cm in diameter on the rat's back.

[0163] The animal grouping information is shown in Table 4 below:

[0164] Table 4. Animal Grouping Information

[0165]

[0166]

[0167] (1) Effect of antibacterial cream on pain in SD rats with deep second-degree burns

[0168] like Figure 27 As shown, a rat model of deep second-degree burns was established by contact burns for 15 seconds.

[0169] like Figure 28 As shown, the antibacterial cream (0.1 g / cm³) was tested using an electronic von Frey analgesic meter. 2 The effect of the antibacterial cream on the mechanical pain threshold of scalded rats was investigated. The model group rats had a significantly lower mechanical pain threshold than the normal group (P < 0.01), while the antibacterial cream group (0.1 g / cm³) had a significantly lower mechanical pain threshold. 2 The mechanical pain threshold of the rats was significantly higher than that of the model group (P < 0.01), indicating that the antibacterial cream (0.1 g / cm³) was effective. 2 It helps to increase the mechanical pain threshold in scalded rats.

[0170] like Figure 29 As shown, the antibacterial cream (0.1 g / cm³) was tested using a Hargreaves pain meter. 2 The effect of the antibacterial cream on the thermal pain threshold of scalded rats was investigated. The results showed that the thermal pain threshold in the model group was significantly lower than that in the normal group (P < 0.01), and the thermal pain threshold in the antibacterial cream group (0.1 g / cm³) was significantly lower. 2 The heat pain threshold in rats was significantly higher than that in the model group (P < 0.01), indicating that the antibacterial cream (0.1 g / cm³) was effective. 2 It helps to increase the thermal pain threshold in scalded rats.

[0171] (2) Effects of antibacterial creams, antibacterial solutions and powders on wound healing in SD rats with deep second-degree burns

[0172] Seven days after administration, the wounds of the animals were photographed and the wound healing rate was calculated. The model group rats had larger wound areas and more exudate. The wound areas of the high-dose antibacterial solution group and the low-dose and high-dose antibacterial cream groups were relatively smaller and had less exudate.

[0173] like Figure 30 , Figure 31 As shown, the wound area of ​​each group of rats was measured and the wound area ratio of each group was compared. It was found that the wound area ratio of the high-dose antibacterial solution group, the low-dose antibacterial cream group, and the high-dose antibacterial cream group was significantly smaller than that of the model group (P<0.05, 0.01), suggesting that the antibacterial solution and antibacterial cream promote wound healing in scalded rats.

[0174] (3) HE staining to observe the effects of antibacterial cream, antibacterial solution and powder on the pathological characteristics of rat wound tissue.

[0175] Three days after administration, wound tissues from rats in each group were collected and subjected to HE staining to observe the pathological changes in the rat wound tissues.

[0176] like Figure 32 As shown, compared with the control group, the epidermis of the model group was thinner or even sloughed off, with partial disappearance of hair follicles and sebaceous glands in the epidermal layer, and a large number of inflammatory cells infiltrated and inflammatory exudate in the subcutaneous layer (indicated by the red arrows). The antibacterial liquid and antibacterial powder groups still showed a large number of inflammatory cells infiltrated and inflammatory exudate in the subcutaneous layer. The antibacterial cream group showed less inflammatory cell infiltration and a higher survival rate of hair follicles and sebaceous glands compared to the antibacterial liquid and antibacterial powder groups.

[0177] like Figure 33 As shown, compared with the model group, the model group showed collagen fiber degeneration and even breakage, with a large number of inflammatory cell infiltrations in the stasis area, necrosis in the epidermis and dermis, and a large number of inflammatory cell infiltrations forming an inflammatory zone between the necrotic tissue and the surviving tissue layer. The antibacterial liquid and antibacterial powder groups still showed a large amount of inflammatory infiltration, and collagen fibers were broken. The antibacterial cream group showed less inflammatory infiltration than the antibacterial liquid and antibacterial powder groups, no obvious collagen fiber breakage, and a shallower inflammatory zone.

[0178] (4) Masson staining was used to observe the effects of antibacterial cream, antibacterial solution and powder on the pathological characteristics of rat wound tissue.

[0179] Three days after administration, wound tissue was collected from rats in each group, and Masson staining was used to observe the pathological changes in the rat wound tissue. Figure 34 (Yellow arrows: damaged sebaceous glands and hair follicles; red arrows: inflammatory cell exudation) As shown, the sebaceous glands and hair follicles in the model group were severely damaged and deformed (yellow arrows), and the collagen was loose. No obvious damage to the sebaceous glands was observed in the drug-treated group, and new collagen had been generated in an orderly manner.

[0180] Seven days after drug administration, wound tissues from rats in each group were collected, and Masson staining was used to observe the pathological changes in the rat wound tissues. Figure 35 As shown, yellow arrows indicate damaged sebaceous glands and hair follicles, black arrows indicate collagen deposition, red arrows indicate inflammatory cell exudation, and green arrows indicate separation of the epidermis and dermis. In the model group, the epidermis and dermis were clearly separated, and the collagen from the inflammatory cell exudation was loosely arranged, while in the drug-treated group, dense new collagen was formed.

[0181] Experiments showed that, using an electronic von Frey pain meter to detect the effect of antibacterial cream on the mechanical pain threshold of scalded rats, the mechanical pain threshold of the model group was significantly lower than that of the normal group (P < 0.01), while the mechanical pain threshold of the antibacterial cream group was significantly higher than that of the model group (P < 0.01), indicating that the antibacterial cream helps to increase the mechanical pain threshold of scalded rats. Using a Hargreaves pain meter to detect the effect of antibacterial cream on the thermal pain threshold of scalded rats, the thermal pain threshold of the model group was significantly lower than that of the normal group (P < 0.01), while the thermal pain threshold of the antibacterial cream group was significantly higher than that of the model group (P < 0.01), indicating that the antibacterial cream helps to increase the thermal pain threshold of scalded rats.

[0182] Seven days after administration, the model group rats had larger wound areas and more exudate. The high-dose antibacterial solution group and the low-dose and high-dose antibacterial cream groups had smaller wound areas and less exudate. Wound areas were measured in each group, and the wound area ratios were compared. The results showed that the wound area ratios in the high-dose antibacterial solution group, the low-dose antibacterial cream group, and the high-dose antibacterial cream group were significantly smaller than those in the model group (P < 0.05, 0.01), suggesting that the antibacterial solution and antibacterial cream promoted wound healing in scalded rats.

[0183] HE staining results showed that in the model group, the epidermis was thinned and even sloughed off, with partial disappearance of hair follicles and sebaceous glands in the epidermis and extensive inflammatory cell infiltration in the subcutaneous layer. In the antibacterial cream group, new hair follicles and sebaceous glands were observed, with more new epidermis and better recovery in both the epidermis and dermis. In the antibacterial liquid and antibacterial powder groups, there was still extensive inflammatory cell infiltration and inflammatory exudation in the subcutaneous layer. Compared with the antibacterial liquid and antibacterial powder groups, the antibacterial cream group had less inflammatory cell infiltration and a higher survival rate of hair follicles and sebaceous glands.

[0184] Masson staining results showed that in the model group, collagen was loose, inflammatory cells were exuded, the epidermis and dermis were severely separated, and sebaceous glands and hair follicles were damaged. After administration, collagen formation in the damaged skin was promoted, inflammatory cells were fewer, and severe sebaceous gland and hair follicle damage was not observed. Specifically, the antibacterial cream group showed significantly more uniform and dense collagen deposition compared to other treatment groups, and fewer inflammatory cells and severely damaged sebaceous glands and hair follicles were observed within the same timeframe. In contrast, the antibacterial powder group showed more inflammatory cell exudation and looser collagen.

[0185] Experimental Example 3

[0186] Clinical trials on burn patients

[0187] Case Study 1: Ms. Li, 53 years old. On the evening of November 30, 2019, she was scalded by boiling water at her workplace. She did not seek medical treatment after the scald, and the wound became red, swollen, and painful. At 5 PM on December 2, she began using the intermediate drug obtained in Example 1 of this invention. The pain disappeared after 15 minutes, and the local redness and swelling at the wound site subsided after 15 hours. On January 7, 2020, the wound healed without leaving a scar. See details... Figure 36 .

[0188] Case Study 2: Lei Mouxing, 34 years old. While inspecting production equipment during a typhoon, 20% of his body was scalded by a jet of 100°C hot water. He was rushed to the People's Hospital for anti-inflammatory and analgesic intravenous treatment. Seven hours later, the intermediate drug obtained in Example 1 of this invention was used; pain relief occurred after 30 minutes, and the entire treatment process was virtually painless. See details... Figure 37 .

[0189] Case Study 3: Li, 16 years old. On January 9, 2021, he suffered burns from the metal outer wall of a boiler. After thirteen days of treatment at a local clinic, the wound ulcerated. On January 23 at 7 PM, the intermediate drug obtained in Example 3 of this invention was used. By 9 AM on January 24, the redness and swelling had subsided, the inflammation was under control, the wound had largely scabbed over, and there was no pain, itching, or pus discharge. On August 1, there was no scar hyperplasia. See details... Figure 38 .

[0190] Case Study 4: He Mouyu, 2 years old. Scalded by boiling water on February 28, 2021, and diagnosed with a 5% deep second-degree burn by Zhejiang Provincial Children's Hospital on the same day. Due to an allergic reaction to cephalosporins, continued medication was not possible. On the third day, the intermediate drug obtained in Example 3 of this invention was started. Pain relief occurred 15 minutes after application, and the entire treatment process was virtually painless. In August 2021, the treatment showed good results in removing necrotic tissue and promoting tissue regeneration, and the patient was essentially cured. See details... Figure 39 .

[0191] Case Study 5: Mr. Ding, 25 years old. On July 23, 2019, he was burned by an electrical spark explosion while conducting an experiment. He was immediately taken to the hospital and received treatment including cooling, pain relief, and antibiotics, as well as topical ointment. After more than a month of hospital treatment, the wound remained painful and itchy, and failed to heal. On August 20, 2019, he began using the intermediate drug obtained in Example 3 of this invention. One and a half hours after application, the erythema and swelling at the wound edges completely subsided. After approximately 20 days of treatment, without the use of alcohol, iodine, or antibiotics, his face peeled three times, and both arms healed completely after drainage and repeated scab formation, leaving no scars. After scab formation at the third-degree burn sites on the webs of his hands, two 3x3cm hypertrophic scars remained. He continued to use traditional Chinese medicine preparations for scar treatment for three months, and the scars completely disappeared. As of August 2021, he was basically healed. See details... Figure 40 .

[0192] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating burns, characterized in that, The raw materials, by weight percentage, are as follows: 40-90% of *Cinnamomum camphora* root, 9-59% of *Cinnamomum cassia* root, and 1-15% of camphor water; the camphor water is prepared by the following method: the branches and leaves of camphor are dried, crushed, and then distilled. The oil-water mixture remaining after distillation, and the aqueous phase after oil-water separation, is the camphor water; the content of dextrorotatory borneol in the camphor water is 0.1-0.5 mg / mL.

2. The traditional Chinese medicine composition for treating burns and scalds according to claim 1, characterized in that, The ingredients, by weight percentage, are as follows: 70% horse thorn root, 20% jade grape root, and 10% camphor water.

3. The traditional Chinese medicine composition for treating burns and scalds according to claim 1, characterized in that, The ingredients, by weight percentage, are as follows: 40% horse thorn root, 59% grape root, and 1% camphor water.

4. The traditional Chinese medicine composition for treating burns according to claim 1, characterized in that, The ingredients, by weight percentage, are as follows: 85% horse thorn root, 10% grape root, and 5% camphor water.

5. A method for preparing the traditional Chinese medicine composition for treating burns and scalds according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Fresh spur roots and grape roots were added to water and preliminarily crushed to obtain liquid I and solid matter after solid-liquid separation; the solid matter was dried and then crushed into powder of 800-1500 mesh; liquid I was separated to obtain liquid II and slurry; finally, the powder and slurry were mixed to obtain a mixture. (2) Add camphor water to the mixture in step (1), mix thoroughly to obtain the original drug intermediate, and refrigerate for later use.

6. The preparation method according to claim 5, characterized in that, The step of progressively crushing the thorn root and the jade grape is as follows: Fresh *Rosa chinensis* or *Vitis thunbergii* roots are added to water and first crushed to 80-100 mesh. Solid-liquid separation is then performed to obtain the liquid. Liquid I and solid matter; after drying, the solid matter is pulverized stepwise at 200 mesh and 800 mesh, and finally pulverized into powder of 800-1500 mesh; liquid I is separated into solid and liquid to obtain liquid II and slurry, the volume of liquid II being 60%-80% of the volume of liquid I; finally, the powder and slurry are mixed to obtain a mixture.

7. The use of the traditional Chinese medicine composition for treating burns according to any one of claims 1-4 in the preparation of a medicament for treating burns.

8. The application according to claim 7, characterized in that, The burn-related diseases mentioned include wound infection, fluid exudation, edema, and pain symptoms caused by burns.

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