Traditional Chinese medicine composition for treating burns and scalds

Abstract

The invention relates to a traditional Chinese medicine composition for treating burns and scalds, which is prepared from spur root and jade grape root according to a certain compatibility, and has the following effects: pain relieving: aching feeling is relieved or even disappears within 15 minutes; convergence: seepage of tissue fluid can be stopped within two hours; bacteriostasis: after the ointment is used, wounds which are just burnt and scalded or fester for several months can be gradually healed, and necrotic tissues can scab and fall off; skin grafting is not needed for burn and scald patients, scar tissue hyperplasia is avoided, and the disability rate of the patients is greatly reduced.

Description

Traditional Chinese medicine composition for treating burns and scalds

[0001] This invention claims priority to the Chinese patent application filed with the China Patent Office on July 21, 2023, with application number 202310902544.6 and invention name “Traditional Chinese Medicine Composition for Treating Burns and Scalds”, the entire contents of which are incorporated by reference into the application. Technical Field

[0002] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for treating burns and scalds. Background Art

[0003] A burn is an injury to the skin or other tissues caused primarily by heat, 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 (scalds).

[0004] Burns can be classified according to the severity of injury as superficial (first-degree burns), superficial partial-thickness burns (superficial second-degree burns), deep partial-thickness burns (deep second-degree burns), and full-thickness burns (third-degree burns). For these non-fatal burns, the wound surface is typically flushed with copious amounts of sterile isotonic saline. If necessary, 0.1-0.2% povidone-iodine (iodine tincture) or 1:2000 chlorhexidine may be used for washing and application. Cold therapy (suitable for mild to moderate burns) can be administered concurrently with debridement. After debridement, exposure or bandaging may be used as appropriate.

[0005] These treatments are often accompanied by severe pain, and even after skin grafting, patients with deep burns and scalds may experience lifelong pain. Burns and scalds can cause extensive interstitial fluid exudation, and those with extensive burns and scalds may even go into shock, a life-threatening condition. Burns and scald wounds that fail to heal are prone to repeated infections and suppuration. Even if the wounds heal, scarring can lead to disfigurement and disability in severe cases. Topical medications can reduce the occurrence of these problems, and therefore play an important role in burn and scald treatment. Studies have shown that traditional Chinese medicine is significantly effective in treating burns.

[0006] For example, patent CN103479917A discloses a Chinese herbal composition for treating burns and scalds, which is prepared from a specific combination of bamboo leaf, Sanguisorba officinalis, Lithospermum officinale, Hempseed Root, Cirsium japonicum, Platycladus orientalis leaves, Achyranthes bidentata, Linderae umbellatae leaves, Rhizoma Coptidis, Coptis chinensis, Snake skin, Red lead, and sesame oil. Another example is patent CN102846778A, which discloses a Chinese herbal medicine for treating burns and scalds, which is prepared from a specific combination of Duchesnea indica, Lobelia chinensis, and Rosa laevigatae leaves, which are washed, dried, crushed, and mixed with an appropriate amount of sesame oil. The composition has anti-inflammatory and analgesic effects, clears away heat and toxins, is astringent and antibacterial, and removes dead tissue and promotes tissue regeneration. It also has the ability to penetrate scabs and eschars, effectively treating burns and scalds without leaving scars. For example, patent CN115444811A discloses a topical borneol camphor oil preparation for treating burns and scalds, and its preparation method. The preparation comprises borneol camphor oil, glycerin, carbomer 940, chitosan, 1,3-butylene glycol, a preservative, a neutralizer, a solubilizing agent, a penetration enhancer, ethanol, and deionized water, using a specific combination. Using borneol camphor oil as the primary ingredient, the preparation is said to improve healing and shorten treatment time. However, existing traditional Chinese medicine formulations have limited efficacy in clinical applications, and the pain, ulceration, and scarring associated with burns remain difficult to address.

[0007] Summary of the Invention

[0008] In response to the deficiencies of the existing technology, the present invention provides a traditional Chinese medicine composition for treating burns and scalds. The composition is prepared from spur root and jade grape root according to a certain combination, so that the traditional Chinese medicine composition has analgesic, astringent, antibacterial and tissue-promoting effects, is highly stable, and can effectively treat burns and scalds without leaving scars.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] A traditional Chinese medicine composition for treating burns and scalds comprises fresh root of Astragalus membranaceus and root of Jade Grape.

[0011] A traditional Chinese medicine composition for treating burns and scalds comprises fresh spur root, jade grape root and camphor water or natural borneol.

[0012] Furthermore, the traditional Chinese medicine composition for treating burns and scalds comprises the following raw materials by weight: 40-90% of spur root, 9-59% of jade grape root and 1-15% of dragon camphor water.

[0013] Preferably, the Chinese medicinal composition for treating burns and scalds comprises the following raw materials by weight: 70% of spur root, 20% of jade grape root and 10% of camphor water.

[0014] Preferably, the Chinese medicinal composition for treating burns and scalds comprises the following raw materials by weight: 40% of spur root, 59% of jade grape root and 1% of camphor water.

[0015] Preferably, the Chinese medicinal composition for treating burns and scalds comprises the following raw materials by weight: 85% of spur root, 10% of jade grape root and 5% of camphor water.

[0016] Preferably, the Chinese medicinal composition for treating burns and scalds comprises the following raw materials by weight: 40-90% of spur root, 10-60% of jasmine root and 1‰-5‰ of natural borneol based on the total amount of spur root and jasmine root.

[0017] Further preferably, the Chinese medicinal composition for treating burns and scalds comprises the following raw materials by weight percentage: 77.8% of Spur Root, 22.2% of Jade Grape Root and 2.5‰ of natural borneol based on the total amount of Spur Root and Jade Grape Root.

[0018] Cirsium chlorolepis Petrak ex Hand.-Mazz is a plant of the Asteraceae family. It can be harvested year-round, but fresh roots harvested in autumn are most effective. Wash and drain the fresh roots, then refrigerate at -20°C until ready for use. Cirsium chlorolepis Petrak ex Hand.-Mazz has blood-cooling, analgesic, antipruritic, antibacterial, tissue-stimulating, and pus-draining properties.

[0019] Jade grape root is derived from the three-lobed snake grape (Ampelopsis delavayana Planch) of the Vitaceae family. Harvested fresh in autumn, it is most effective. Wash, drain, and core the fresh roots, then refrigerate at -20°C until ready to use. Jade grape root has analgesic, antiseptic, detoxifying, and anti-inflammatory properties.

[0020] In the traditional Chinese medicine composition for treating burns and scalds of the present invention, the borneol camphor water is prepared by the following method: branches and leaves of borneol camphor are dried, crushed and then distilled, and an oil-water mixture remains after distillation. The water phase after oil-water separation is the borneol camphor water.

[0021] Furthermore, the content of D-borneol in the camphor water is 0.1-0.5 mg / mL.

[0022] The method for preparing the above-mentioned Chinese medicine composition for treating burns and scalds comprises the following steps:

[0023] (1) Fresh spur root and jade grape root are added with water, crushed, and then solid-liquid separation is performed to obtain liquid I and solid; the solid is dried and crushed into a powder of 800-1500 mesh; liquid I is solid-liquid separated to obtain liquid II and a slurry; finally, the powder and the slurry are mixed to obtain a mixture;

[0024] (2) Adding borneol water or natural borneol to the mixture of step (1), mixing thoroughly to obtain the original drug intermediate, and refrigerating for later use.

[0025] Furthermore, in step (1), the step of stepwise crushing is as follows: fresh spur root or jade grape root is added with water, first crushed to 80-100 mesh, and solid-liquid separation is performed to obtain liquid I and solid; after the solid is dried, it is crushed stepwise according to 200 mesh and 800 mesh, and finally crushed to 800-1500 mesh powder; liquid I is separated into 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.

[0026] Furthermore, in step (1), the amount of water added is 2-10 times the mass of fresh spur root or jade grape root.

[0027] Furthermore, in step (2), the refrigerated temperature is 4-8°C, preferably 4°C.

[0028] The above-mentioned Chinese medicinal composition for treating burns and scalds is used in the preparation of medicines for treating burn and scald diseases, wherein the burn and scald diseases include wound infection, fluid exudation, edema and pain symptoms caused by burns and scalds.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention uses fresh horsetail root and jade grape root in a certain combination to prepare a Chinese medicine composition for treating burns and scalds. The fresh horsetail root and jade grape root work together to make the Chinese medicine composition have the effects of analgesia: the pain is relieved or even disappears within fifteen minutes, and the use of analgesics can be stopped; astringency: the exudation of tissue fluid can be stopped within two hours; antibacterial: the wounds of fresh burns and scalds or wounds that have been ulcerated for several months can gradually heal after use, and the necrotic tissue will scab and fall off; and muscle regeneration: patients with burns and scalds do not need skin grafting, and scar tissue will not proliferate, which greatly reduces the disability rate of patients.

[0031] (2) The Chinese medicine composition of the present invention also has good stability.

[0032] (3) The borneol water of the present invention is the aqueous solution remaining after the branches and leaves of borneol camphor are extracted and processed into natural borneol. The addition of borneol camphor water can enhance the healing effect of the affected area, shorten the treatment time, and improve stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 shows the effect of antibacterial cream on HaCaT cells observed under a light microscope;

[0034] FIG2 is an observation of the effect of the antibacterial solution on HaCaT cells under a light microscope;

[0035] Figure 3 shows the effect of antibacterial cream on HaCaT cell migration;

[0036] FIG4 shows the effect of the antibacterial solution on HaCaT cell migration;

[0037] FIG5 is the effect of the antibacterial solution on HaCaT cell apoptosis observed by TUNEL staining;

[0038] FIG6 is the effect of the antibacterial solution on HaCaT cell proliferation observed by CCK8 method;

[0039] FIG7 is the effect of antibacterial cream on HaCaT cell aging observed by β-gal staining;

[0040] FIG8 is the effect of the antibacterial solution on HaCaT cell senescence observed by β-gal staining;

[0041] FIG9 is an observation of the effect of antibacterial cream on HFF cells under a light microscope;

[0042] FIG10 is an observation of the effect of the antibacterial solution on HFF cells under a light microscope;

[0043] Figure 11 shows the effect of antibacterial cream on HFF cell migration;

[0044] FIG12 shows the effect of the antibacterial solution on HFF cell migration;

[0045] FIG13 is a flow cytometry analysis of the effect of the antibacterial solution on HFF cell apoptosis;

[0046] FIG14 is the effect of antibacterial cream on HFF cell apoptosis observed by TUNEL staining;

[0047] FIG15 is the effect of the antibacterial solution on HFF cell apoptosis observed by TUNEL staining;

[0048] FIG16 is the effect of the antibacterial solution on HFF cell proliferation observed by CCK8 method;

[0049] FIG17 is the effect of antibacterial cream on HFF cell senescence observed by β-gal staining;

[0050] FIG18 is the effect of the antibacterial solution on HFF cell senescence observed by β-gal staining;

[0051] FIG19 is an observation of the effect of antibacterial cream on HUVEC cells under a light microscope;

[0052] FIG20 is an observation of the effect of the antibacterial solution on HUVEC cells under a light microscope;

[0053] FIG21 shows the effect of antibacterial cream on HUVEC cell migration;

[0054] FIG22 shows the effect of antibacterial solution on HUVEC cell migration;

[0055] FIG23 is a flow cytometry analysis of the effect of the antibacterial solution on HUVEC cell apoptosis;

[0056] FIG24 is the effect of the antibacterial solution on HUVEC cell proliferation observed by CCK8 method;

[0057] FIG25 is the effect of antibacterial cream on HUVEC cell senescence observed by β-gal staining;

[0058] FIG26 is the effect of the antibacterial solution on HUVEC cell senescence observed by β-gal staining;

[0059] FIG27 shows the establishment of a rat deep second-degree burn model;

[0060] FIG28 shows the effect of antibacterial cream on the mechanical pain threshold of rats with deep second-degree scald, wherein N=6, **, P<0.01 compared with the normal group; ##, P<0.01 compared with the model group;

[0061] FIG29 shows the effect of antibacterial cream on the thermal pain threshold of rats with deep second-degree scald, wherein N=4, **, P<0.01 compared with the normal group; ##, P<0.01 compared with the model group;

[0062] Figure 30 is a photograph 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;

[0063] Figure 31 shows the effects of antibacterial liquid, antibacterial cream and powder on the burn area of ​​deep II degree burns in rats (administered for 7 days) **, P < 0.01, *, P < 0.05 compared with the model group;

[0064] Figure 32 shows the pathological changes of rat skin wound tissue observed by HE staining (administered for 3 days), where A. 4×, B. 10×;

[0065] Figure 33 shows the pathological changes of rat skin wound tissue observed by HE staining (7 days after administration), where A. 4×, B. 10×;

[0066] Figure 34 shows the pathological changes of rat skin wound tissue observed by Masson staining (3 days of drug administration), where A. 4×, B. 10×; yellow arrows: damaged sebaceous glands and hair follicles, red arrows: inflammatory cell infiltration;

[0067] Figure 35 shows the pathological changes of rat skin wound tissue observed by Masson staining (7 days of drug administration), where A. 4×, B. 10×; yellow arrows: damaged sebaceous glands and hair follicles, black arrows: collagen deposition, red arrows: inflammatory cell infiltration, green arrows: separation of the epidermis and dermis;

[0068] FIG36 is an injury diagram of typical case 1 of the present invention;

[0069] FIG37 is an injury diagram of typical case 2 of the present invention;

[0070] FIG38 is an injury diagram of typical case 3 of the present invention;

[0071] FIG39 is an injury diagram of typical case 4 of the present invention;

[0072] FIG40 is an injury diagram of typical case 5 of the present invention. DETAILED DESCRIPTION

[0073] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following is merely an illustrative description of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0074] Unless otherwise specified, the various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels.

[0075] Example 1

[0076] A traditional Chinese medicine composition for treating burns and scalds is prepared by the following method: 700g of fresh spur root and 200g of jasmine grape root are mixed with 4 wt% of water, and the mixture is first crushed to 100 mesh, followed by solid-liquid separation to obtain a solid and a liquid I, wherein the mesh size of particles contained in the liquid I is greater than 800 mesh; after drying, the solid is crushed in steps of 200 mesh and 800 mesh, and finally crushed to a powder of 1000 mesh; the liquid I is further subjected to solid-liquid separation to obtain a liquid II and a slurry, wherein the volume of the liquid II is 70% of the volume of the liquid I; the powder and the slurry are mixed, and 100g of cinnamon bark water is added simultaneously, and the mixture is thoroughly mixed to obtain a technical intermediate, which is then refrigerated at 4°C for later use.

[0077] The preparation method of camphor water is as follows: the branches and leaves of camphor tree are dried, crushed and then distilled, and an oil-water mixture remains after distillation. The water phase after the oil and water are separated is the camphor water.

[0078] Example 2

[0079] A traditional Chinese medicine composition for treating burns and scalds is prepared by the following method: 400g of fresh root of horsetail and 590g of root of grapevine are respectively mixed with 4 wt% of water, firstly crushed to 100 mesh, and solid-liquid separation is performed to obtain a solid and liquid I, wherein the mesh size of particles contained in liquid I is greater than 800 mesh; after drying, the solid is crushed stepwise according to 200 mesh and 800 mesh, and finally crushed to a powder of 1000 mesh; liquid I is solid-liquid separation to obtain liquid II and a slurry, wherein the volume of liquid II is 68% of the volume of liquid I; the powder and the slurry are mixed, and 10g of cyperus rotundus water is added simultaneously. The preparation method of cyperus rotundus water is the same as that of Example 1. After thorough mixing, a raw material intermediate is obtained, and the raw material is refrigerated at 4°C for later use.

[0080] Example 3

[0081] A traditional Chinese medicine composition for treating burns and scalds is prepared by the following method: 850g of fresh spur root and 100g of jasmine root are mixed with 4 wt% of water, and the mixture is first crushed to 100 mesh, followed by solid-liquid separation to obtain a solid and a liquid I, wherein the mesh size of particles contained in the liquid I is greater than 800 mesh; after drying, the solid is crushed in steps of 200 mesh and 800 mesh, and finally crushed to a powder of 1000 mesh; the liquid I is subjected to solid-liquid separation to obtain a liquid II and a slurry, wherein the volume of the liquid II is 76% of the volume of the liquid I; the powder and the slurry are mixed, and 50g of cyperus rotundus water is added simultaneously. The cyperus rotundus water is prepared in the same manner as in Example 1, and after thorough mixing, a raw material intermediate is obtained, which is refrigerated at 4°C for later use.

[0082] Example 4

[0083] The difference from Example 1 is that the dragon camphor water is replaced by natural borneol, and the rest is the same as Example 1. Specifically, a traditional Chinese medicine composition for treating burns and scalds is prepared by the following method: 700g of fresh spur root and 200g of jade grape root are mixed with 4 weight of water, first crushed to 100 mesh, and solid-liquid separation is performed to obtain a solid and liquid I, wherein the mesh size of the particles contained in liquid I is greater than 800 mesh, and after the solid is dried, it is crushed step by step according to 200 mesh and 800 mesh, and finally crushed to a powder of 1000 mesh; liquid I is further solid-liquid separated to obtain liquid II and a slurry, and the volume of liquid II is 70% of the volume of liquid I; the powder and the slurry are mixed, and 2.5‰ natural borneol is added to the mixture by weight, and after thorough mixing, a raw drug intermediate is obtained, and refrigerated at 4°C for use.

[0084] Example 5

[0085] Preparation of antibacterial solution: The preparation method of the antibacterial solution is conventional technology, that is, the original drug intermediate of Example 1 is used, purified water and sodium benzoate are added to obtain the antibacterial solution.

[0086] Example 6

[0087] Preparation of antibacterial cream. The preparation method of antibacterial cream is also conventional technology. The original drug intermediate of Example 1 is used, and carbomer and sodium benzoate are added to obtain antibacterial cream.

[0088] Example 7

[0089] Preparation of antibacterial powder: The preparation method of antibacterial powder is also conventional technology, using the original drug intermediate of Example 1, drying and then crushing to obtain antibacterial powder.

[0090] Comparative Examples 1-4

[0091] Comparative Examples 1-4 differ from Example 1 in that, in the comparative examples, the root of Jade Grape was replaced with Seven Leaf Lotus, White Ampelopsis, Rosa Laevigata Fruit, and Rubia cordifolia, respectively; otherwise, the same as in Example 1. The technical intermediates prepared in Example 1 and the comparative examples were used to treat burns and scalds. Each group had at least 50 cumulative users, and the efficacy was scored based on analgesia, astringency, antibacterial activity, and myogenicity. Example 1 was given a full score of 5, and higher scores for the other compounds indicated better efficacy. The results are shown in Table 1.

[0092] Table 1.

[0093] Test 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 the antibacterial solution and antibacterial cream on cell morphology were observed under a light microscope. The cell scratch test was used to observe the effects of the antibacterial solution and antibacterial cream on cell migration ability. The flow cytometry and TUNEL staining were used to observe the effects of the antibacterial solution and antibacterial cream on cell apoptosis. The CCK8 method was used to detect the effects of the antibacterial solution and antibacterial cream on cell proliferation. The β-gal staining method was used to observe the effects of the antibacterial solution and antibacterial cream on cell senescence.

[0096] The cells included the following three types: epidermal cells: human immortalized keratinocyte line (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); 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 frozen cell tube and shake it in a 37°C water bath to rapidly thaw. Spray the outside of the tube thoroughly with alcohol for disinfection and transfer it to a biosafety cabinet. Transfer the cell suspension to a 15mL centrifuge tube and slowly add complete culture medium dropwise until the volume reaches 5-6mL. Secure the lid and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and retain the cell pellet. Add an appropriate amount of complete culture medium to the centrifuge tube. Gently pipette the cell pellet 7-8 times to prepare a uniform cell suspension. Transfer the suspension to a culture dish and culture in a cell incubator. Observe cell attachment and growth regularly.

[0099] Cell passaging: When the cells grow to a confluency greater than 70%-80%, they should be passaged immediately. After discarding the supernatant, rinse the cells with an appropriate amount of PBS to remove dead cells and residual serum, then add trypsin to digest for about 30 seconds before adding serum to terminate the digestion. Gently pipette the cells into a uniform single cell suspension and transfer them to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and retain the cell pellet. Then add an appropriate amount of complete culture medium to make a cell suspension. Add it to a new cell culture dish according to the experimental requirements, culture it in a cell culture incubator, and regularly observe the cell adhesion and growth to complete the corresponding subsequent experiments.

[0100] Flow cytometry was used to detect cell apoptosis

[0101] Seed cells in a six-well plate, add drug, aspirate the medium from each well, add pre-chilled PBS, gently shake the six-well plate to wash away floating cells, add Accutase solution, and incubate in an incubator for 1-2 minutes. Once the cells have completely floated, add serum to neutralize and terminate the digestion. Collect the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Wash the cell pellet 2-3 times with PBS. Resuspend the cells in 1x Binding Buffer diluted to 500 μL, then add 5 μL of Annexin V-APC and 10 μL of 7-AAD. Gently vortex to mix and incubate at room temperature in the dark for 5 minutes. Detect Annexin V-APC using the APC detection channel and 7-AAD using the PI detection channel on a flow cytometer.

[0102] Cell scratch assay

[0103] First, prepare a sterile 6-well plate and evenly draw lines on the bottom of each well with a marker pen, drawing three lines in each well with a distance of 1 cm between each line. Then, inoculate cells in the 6-well plate and add the drug to be tested. When the cell confluence reaches more than 90%, use a 200μL pipette tip perpendicular to the bottom of the well to evenly and forcefully cut the cells along the line drawn by the marker pen. Rinse the detached cells with PBS, then add 2mL of culture medium containing 0.5% FBS to each well. Observe under a microscope and take pictures to record the cell status around the line, which is recorded as the cell position at 0h. Continue to incubate the 6-well plate and take pictures of the cell position after 24h.

[0104] CCK8 assay to detect cell proliferation

[0105] Seed cells in a 96-well plate at 5,000 cells per well and incubate in a cell culture incubator for 4-6 hours. After the cells have attached, add 10 μL of CCK-8 solution to each well. Incubate in the cell culture incubator for another 2 hours. Measure the absorbance at 450 nm using a microplate reader and record the data. This represents the cell 0 hour. Subsequently, measure the absorbance at 6, 12, and 24 hours of incubation.

[0106] Beta-galactosidase staining (β-gal staining)

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

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

[0109] TUNEL staining to detect cell apoptosis

[0110] Wash cells once with PBS, fix them with 4% paraformaldehyde for 30 minutes, wash once with PBS, add PBS containing 0.3% Triton X-100, and incubate at room temperature for 5 minutes. Wash twice with PBS, add 50 μL of TUNEL detection solution (preparation method see Table 3) to the sample, incubate at 37°C in the dark for 60 minutes, wash three times with PBS, mount the slides with anti-fluorescence quenching solution, and observe under a fluorescence microscope.

[0111] Table 3. Preparation of TUNEL detection solution

[0112] (1) Effects of antibacterial liquid and antibacterial cream on epidermal cells

[0113] a. Observe the effects of antibacterial liquid and antibacterial cream on epidermal cells under light microscope

[0114] After adding different concentrations of antibacterial cream to HaCaT cells cultured in vitro, some cells underwent transformation and became fibroblasts (Figure 1, indicated by red arrows), suggesting that epidermal cells may have undergone epithelial-mesenchymal cell transformation.

[0115] After adding different concentrations of antibacterial solution to HaCaT cells cultured in vitro, some cells also underwent transformation (Figure 2, indicated by red arrows), suggesting that epidermal cells may have undergone epithelial-mesenchymal cell transformation.

[0116] b. Effects of antibacterial cream and antibacterial solution on epidermal cell migration

[0117] The effect of the antibacterial cream on epidermal cell migration was observed using a scratch test. As shown in Figure 3, after 24 hours of antibacterial cream treatment, the intercellular spaces between cells were smaller when the 0.05 mg / mL antibacterial cream was applied, suggesting that the antibacterial cream promoted epidermal cell migration.

[0118] The effect of the antibacterial solution on epidermal cell migration was observed using a scratch test. As shown in Figure 4, after 24 hours of antibacterial solution treatment, the intercellular spaces between the 50 μL / mL and 100 μL / mL groups were smaller, suggesting that the antibacterial solution promoted epidermal cell migration.

[0119] c. Effects of antibacterial cream and antibacterial solution on epidermal cell apoptosis

[0120] The effect of the antibacterial solution on epidermal cell apoptosis was observed by TUNEL staining. As shown in Figure 5 , there was no significant change in the apoptosis rate of cells in each group.

[0121] d. Effect of antibacterial solution on epidermal cell proliferation

[0122] The CCK8 method was used to detect the effect of the antibacterial solution on the proliferation of epidermal cells, and it was found that different concentrations of antibacterial solution could promote the proliferation of epidermal cells (Figure 6).

[0123] e. Effects of antibacterial liquid and antibacterial cream on epidermal cell aging

[0124] The β-gal staining method was used to observe the effect of antibacterial cream on epidermal cell aging. No increase in the number of stained epidermal cells was found, suggesting that the antibacterial cream may not affect the aging of epidermal cells (Figure 7).

[0125] The β-gal staining method was used to observe the effect of the antibacterial solution on epidermal cell senescence. No increase in the stained epidermal cells was found, suggesting that the antibacterial solution may not affect the senescence of epidermal cells ( Figure 8 ).

[0126] (2) Effects of antibacterial liquid and antibacterial cream on fibroblasts

[0127] a. Observe the effects of antibacterial liquid and antibacterial cream on fibroblasts under light microscope

[0128] After adding antibacterial cream to HFF cells cultured in vitro, no obvious changes occurred in the cells (Figure 9).

[0129] After adding the antibacterial solution to HFF cells cultured in vitro, no obvious changes occurred in the cells (Figure 10).

[0130] b. Effects of antibacterial cream and antibacterial liquid on fibroblast migration

[0131] The effect of the antibacterial cream on fibroblast migration was observed using a scratch test. As shown in Figure 11, after 24 hours of antibacterial cream treatment, the intercellular spaces in the 0.05 mg / mL antibacterial cream group were smaller, suggesting that the antibacterial cream promoted fibroblast migration.

[0132] The effect of the antibacterial solution on fibroblast migration was observed by a scratch test. As shown in Figure 12, after 24 hours of antibacterial solution treatment, the intercellular spaces in the 50 μL / mL and 100 μL / mL antibacterial solution groups were smaller, indicating that the antibacterial solution promoted the migration of fibroblasts.

[0133] c. Effects of antibacterial liquid and cream on apoptosis of fibroblasts

[0134] The effect of the antibacterial solution on fibroblast apoptosis was observed by flow cytometry. As shown in FIG13 , there was no significant change in the apoptosis rate of cells in each group, indicating that the antibacterial solution may have no effect on the apoptosis of fibroblasts.

[0135] The effect of the antibacterial cream on fibroblast apoptosis was observed by TUNEL staining. As shown in FIG14 , there was no significant change in the apoptosis rate of cells in each group, suggesting that the antibacterial cream may have no effect on the apoptosis of fibroblasts.

[0136] The effect of the antibacterial solution on fibroblast apoptosis was observed by TUNEL staining. As shown in FIG15 , there was no significant change in the apoptosis rate of cells in each group, indicating that the antibacterial solution may have no effect on the apoptosis of fibroblasts.

[0137] c. Effect of antibacterial solution on fibroblast proliferation

[0138] The CCK8 method was used to detect the effect of the antibacterial solution on the proliferation of fibroblasts, and it was found that different concentrations of the antibacterial solution could promote the proliferation of fibroblasts ( Figure 16 ).

[0139] d. Effects of antibacterial liquid and antibacterial cream on fibroblast senescence

[0140] The β-gal staining method was used to observe the effect of antibacterial cream on fibroblast senescence. No increase in the number of stained fibroblasts was found, suggesting that the antibacterial cream may not affect the senescence of fibroblasts ( Figure 17 ).

[0141] The β-gal staining method was used to observe the effect of the antibacterial solution on fibroblast senescence. No increase in the number of stained fibroblasts was found, suggesting that the antibacterial solution may not affect the senescence of fibroblasts ( Figure 18 ).

[0142] (3) Effects of antibacterial liquid and antibacterial cream on endothelial cells

[0143] a. Observe the effects of antibacterial liquid and antibacterial cream on endothelial cells under light microscope

[0144] After adding the antibacterial cream to HUVEC cells cultured in vitro, the number of HUVEC cells decreased after the action of high concentration of the antibacterial cream ( FIG. 19 ).

[0145] After adding the antibacterial solution to HUVEC cells cultured in vitro, the number of HUVEC cells decreased after the action of high concentration of the antibacterial solution ( FIG. 20 ).

[0146] b. Effects of antibacterial cream and antibacterial liquid on endothelial cell migration

[0147] The effect of antibacterial cream on HUVEC cell migration was observed by scratch test. As shown in Figure 21, after 24 hours of antibacterial cream treatment, the intercellular gaps in the 0.10 mg / mL antibacterial cream group were smaller, indicating that the antibacterial cream promoted the migration of HUVEC cells.

[0148] The effect of the antibacterial solution on endothelial cell migration was observed by a scratch test. As shown in FIG22 , after 24 hours of the antibacterial solution treatment, the intercellular gaps between the groups were relatively small, indicating that the antibacterial solution had a weak effect on endothelial cell migration.

[0149] c. Effects of antibacterial liquid and antibacterial cream on endothelial cell apoptosis

[0150] The effect of the antibacterial solution on endothelial cell apoptosis was observed by flow cytometry. As shown in FIG23 , there was no significant change in the apoptosis rate of cells in each group.

[0151] d. Effects of antibacterial solution on endothelial cell proliferation

[0152] The CCK8 method was used to detect the effect of the antibacterial solution on endothelial cell proliferation, and it was found that different concentrations of the antibacterial solution could promote the proliferation of endothelial cells ( Figure 24 ).

[0153] e. Effects of antibacterial liquid and antibacterial cream on endothelial cell aging

[0154] The β-gal staining method was used to observe the effect of antibacterial cream on endothelial cell senescence. No increase in the stained endothelial cells was found, suggesting that the antibacterial cream may not affect the senescence of endothelial cells (Figure 25).

[0155] The β-gal staining method was used to observe the effect of the antibacterial solution on endothelial cell senescence. No increase in the stained endothelial cells was found, suggesting that the antibacterial solution may not affect the senescence of endothelial cells ( Figure 26 ).

[0156] Experiments have shown that antibacterial liquid and antibacterial cream can promote the transformation and migration of epidermal cells, and help promote the migration of new skin flaps during burn recovery; antibacterial liquid and antibacterial cream promote the proliferation of fibroblasts, and help promote granulation tissue formation on burn wounds.

[0157] Experimental Example 2

[0158] 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

[0159] Positive control drug information, sulfadiazine silver cream, specifications 500g: 5g, manufacturer: Guangdong Hengjian Pharmaceutical Co., Ltd.

[0160] Experimental Animals and Models: SD rats (200-220 g, male) were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. After 1 week of acclimatization, the rats were randomly divided into groups. 5% sodium sulfide was applied to the back of the rats for hair removal. The rats were anesthetized with small animal anesthetic and were exposed to a 2.5 cm diameter metal object for 15 seconds, resulting in a circular or oval deep II wound approximately 2 cm in diameter on the back of the rats.

[0161] The animal grouping information is as described in Table 4 below:

[0162] Table 4. Animal group information

[0163] (1) Effect of antibacterial cream on pain in SD rats with deep second-degree burns

[0164] As shown in FIG27 , a deep second-degree burn model in rats was established by contact burn for 15 seconds.

[0165] As shown in Figure 28, the antibacterial cream (0.1 g / cm 2 ) on the mechanical pain threshold of scalded rats. It was found that the mechanical pain threshold of rats in the model group was significantly lower than that in the normal group (P<0.01), and the antibacterial cream group (0.1g / cm 2 ) rats had a significantly higher mechanical pain threshold than the model group (P<0.01), indicating that the antibacterial cream (0.1 g / cm 2 ) helps to increase the mechanical pain threshold of scalded rats.

[0166] As shown in Figure 29, the Hargreaves pain meter was used to measure the antibacterial cream (0.1 g / cm 2) on the thermal pain threshold of scalded rats. It was found that the thermal pain threshold of the rats in the model group was significantly lower than that in the normal group (P<0.01), and the thermal pain threshold of the rats in the antibacterial cream group (0.1g / cm 2 ) rats' thermal pain threshold was significantly higher than that of the model group (P<0.01), indicating that the antibacterial cream (0.1 g / cm 2 ) helps to increase the thermal pain threshold of scalded rats.

[0167] (2) Effects of antibacterial cream, antibacterial liquid, and powder on wound healing in SD rats with deep second-degree burns

[0168] Seven days after administration, the wounds of the animals were photographed and the wound healing rates were calculated. The wounds of the rats in the model group were larger and had more exudate. The wounds of the rats in the high-dose antibacterial liquid group and the low-dose and high-dose antibacterial cream groups were relatively smaller and had less exudate.

[0169] As shown in Figures 30 and 31, the wound areas of rats in each group were measured respectively, and the wound area ratios of each group were compared. It was found that the wound area ratios of the high-dose antibacterial liquid group, the low-dose antibacterial cream group, and the high-dose antibacterial cream group were significantly smaller than that of the model group (P<0.05, 0.01), indicating that the antibacterial liquid and antibacterial cream promoted wound healing in scalded rats.

[0170] (3) HE staining to observe the effects of antibacterial cream, antibacterial liquid and powder on rat wound tissue pathology

[0171] Three days after administration, the wound tissues of the rats in each group were collected and stained with HE to observe the pathological changes of the wound tissues of the rats.

[0172] As shown in Figure 32, compared with the control group, the epidermis in the model group became thinner and even shed, the hair follicles and sebaceous glands in the epidermis partially disappeared, and a large number of inflammatory cells infiltrated subcutaneously, with inflammatory exudates (indicated by the red arrows). The antibacterial liquid and antibacterial powder groups still had a large number of inflammatory cells infiltrating subcutaneously, and inflammatory exudates were still present. The antibacterial cream group had less inflammatory cell infiltration than the antibacterial liquid and antibacterial powder groups, and a higher survival rate of hair follicles and sebaceous glands.

[0173] As shown in Figure 33, compared with the model group, the model group showed collagen fiber degeneration and even rupture. Numerous inflammatory cell infiltration was observed in the stasis zone. Necrosis occurred in the epidermis and dermis, and a large number of inflammatory cells infiltrated between the necrotic and viable tissue layers, forming an inflammatory zone. The antibacterial liquid and antibacterial powder groups still showed extensive inflammatory infiltration and collagen fiber rupture. The antibacterial cream group showed less inflammatory infiltration than the antibacterial liquid and antibacterial powder groups, no obvious collagen fiber rupture, and a shallower inflammatory zone.

[0174] (4) Masson staining to observe the effects of antibacterial cream, antibacterial liquid and powder on rat wound tissue pathology

[0175] Three days after administration, wound tissue from each group was obtained and Masson staining was performed to observe pathological changes in the rat wounds. As shown in Figure 34 (yellow arrows: damaged sebaceous glands and hair follicles; red arrows: inflammatory cell infiltration), the sebaceous glands and hair follicles in the model group were severely damaged and deformed (yellow arrows), with loose collagen. In the treatment group, no obvious damage to the sebaceous glands was observed, and neatly arranged new collagen had been generated.

[0176] Seven days after administration, wound tissue from each group was obtained and Masson staining was performed to observe pathological changes in the rat wounds. As shown in Figure 35, 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, with loosely arranged collagen due to inflammatory cell exudation, while in the treatment group, dense new collagen was formed.

[0177] The experiment showed that the effect of antibacterial cream on the mechanical pain threshold of scalded rats was detected using an electronic von Frey analgesia. The mechanical pain threshold of rats in the model group was significantly lower than that in the normal group (P<0.01), while the mechanical pain threshold of rats in the antibacterial cream group was significantly higher than that in the model group (P<0.01), indicating that antibacterial cream can help increase the mechanical pain threshold of scalded rats. The effect of antibacterial cream on the thermal pain threshold of scalded rats was detected using a Hargreaves analgesia. The thermal pain threshold of rats in the model group was significantly lower than that in the normal group (P<0.01), while the thermal pain threshold of rats in the antibacterial cream group was significantly higher than that in the model group (P<0.01), indicating that antibacterial cream can help increase the thermal pain threshold of scalded rats.

[0178] Seven days after administration, the model group had larger wound areas and more exudate, while the high-dose antibacterial solution group and the low- and high-dose antibacterial cream groups had smaller wound areas and less exudate. Wound area measurements were performed on each group and the wound area ratios were compared. The high-dose antibacterial solution group, low-dose antibacterial cream group, and high-dose antibacterial cream group showed significantly smaller wound area ratios than the model group (P < 0.05, 0.01), suggesting that the antibacterial solution and antibacterial cream promote wound healing in scalded rats.

[0179] HE staining results showed that the epidermis in the model group thinned and even shed, with partial disappearance of epidermal hair follicles and sebaceous glands, and a large number of inflammatory cells infiltrating the subcutaneous layer. In the antibacterial cream group, hair follicles and sebaceous glands were newly formed, with more newly formed epidermis, and both the epidermis and dermis recovered well. In the antibacterial liquid and antibacterial powder groups, a large number of inflammatory cells infiltrated the subcutaneous layer, and inflammatory exudation was still present. 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.

[0180] Masson staining revealed that the model group exhibited loose collagen, inflammatory cell infiltration, severe separation between the epidermis and dermis, and damage to the sebaceous glands and hair follicles. Drug administration promoted collagen formation in the damaged skin, reduced inflammatory cells, and prevented severe damage to the sebaceous glands and hair follicles. The antibacterial cream group exhibited significantly more uniform and dense collagen deposition compared to the other treatment groups, with less inflammatory cell infiltration and severely damaged sebaceous glands and hair follicles over the same time period. The antibacterial powder group, on the other hand, exhibited greater inflammatory cell infiltration and looser collagen.

[0181] Experimental Example 3

[0182] Clinical trials on patients with burns and scalds

[0183] Case Study 1: Li Mouping, 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 and experienced redness, swelling, and pain at the wound. At 5:00 PM on December 2, she began using the technical intermediate obtained in Example 1. The pain subsided after 15 minutes, and the local redness and swelling at the wound site subsided 15 hours later. By January 7, 2020, the wound had healed, and she recovered without scarring. See Figure 36 for details.

[0184] Case 2: Lei Mouxing, 34 years old. While inspecting production equipment during a typhoon, he sustained burns covering 20% ​​of his body from 100°C hot water. He was rushed to the People's Hospital and received anti-inflammatory and analgesic infusions. Seven hours later, he administered the technical intermediate obtained in Example 1 of the present invention. The pain subsided within 30 minutes, and he experienced virtually no pain throughout the entire treatment. See Figure 37 for details.

[0185] Case Study 3: Li, 16 years old. On January 9, 2021, he suffered burns from the outer metal wall of a boiler. After thirteen days of treatment at a local clinic, the wound ulcerated. At 7 PM on January 23, he used the technical intermediate obtained in Example 3 of this invention. By 9 AM on January 24, the redness and swelling had subsided, the inflammation was under control, and the wound had essentially scabbed over. The wound was painless, itchy, and pus-free. By August 1, the wound had no scarring. See Figure 38 for details.

[0186] Case 4: He Mouyu, 2 years old. On February 28, 2021, he was scalded by boiling water and diagnosed with a 5% deep second-degree burn by the Zhejiang Provincial Children's Health Care Center that same day. He was unable to continue treatment due to a cephalosporin allergy. On the third day, he began using the technical intermediate obtained in Example 3 of the present invention. Pain relief occurred 15 minutes after application, and the entire treatment process was virtually painless. By August 2021, the patient had achieved excellent results in removing dead tissue and promoting muscle regeneration, and was essentially fully recovered. See Figure 39 for details.

[0187] Typical case 5: Ding, 25 years old. On July 23, 2019, he was burned by an electric spark explosion while doing an experiment. He was immediately sent to the hospital for cooling, analgesic and antibiotic treatment, and applied external plasters. After more than a month of treatment in the hospital, the wound was painful and itchy and could not heal. On August 20, 2019, he started to use the original drug intermediate obtained in Example 3 of the present invention. One and a half hours after the medication, the erythema and swelling at the edge of the wound subsided. After about 20 days of treatment, no alcohol, iodine tincture or antibiotics were used during the treatment period. The face was peeled three times, and the arms were healed after pus drainage and repeated scabbing without leaving scars. After the scabs formed on the third-degree burns on the base of the palms of both hands, there were two 3X3CM hypertrophic scars. After continuing to use Chinese medicine preparations for anti-scar treatment for three months, the scars were completely eliminated. The current situation in August 2021 is basically healed. See Figure 40 for details.

[0188] The above description of the embodiments is intended to facilitate understanding and application of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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 present invention, should be within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for treating burns and scalds, characterized in that: The prescription contains: spur root and jade grape root.

2. The Chinese medicine composition for treating burns and scalds according to claim 1, characterized in that: Calculated by weight percentage, the invention comprises the following raw materials: 40-90% of spur root, 9-59% of jade grape root and 1-15% of camphor water.

3. The Chinese medicine composition for treating burns and scalds according to claim 2, characterized in that: Calculated by weight percentage, the invention comprises the following raw materials: 70% of spur root, 20% of jade grape root and 10% of dragon camphor water.

4. The Chinese medicine composition for treating burns and scalds according to claim 2, characterized in that: Calculated by weight percentage, the composition comprises the following raw materials: 40% of spur root, 59% of jade grape root and 1% of camphor water.

5. The Chinese medicine composition for treating burns and scalds according to claim 2, characterized in that: The invention comprises the following raw materials by weight percentage: 85% of spur root, 10% of jade grape root and 5% of camphor water.

6. The Chinese medicine composition for treating burns and scalds according to claim 1, characterized in that: The raw materials are as follows: 40-90% of spur root, 10-60% of jade grape root and 1‰-5‰ of natural borneol based on the total amount of spur root and jade grape root.

7. The Chinese medicine composition for treating burns and scalds according to claim 6, characterized in that: Calculated by weight percentage, the raw materials include: 77.8% of spur root, 22.2% of jade grape root and 2.5‰ of the auxiliary material natural borneol of the total amount of spur root and jade grape root.

8. The Chinese medicine composition for treating burns and scalds according to any one of claims 1 to 5, characterized in that: The camphor water is prepared by the following method: branches and leaves of camphor are dried, crushed and then distilled, and an oil-water mixture remains after distillation. The water 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.

9. The method for preparing the Chinese medicine composition for treating burns and scalds according to any one of claims 1 to 8, characterized in that: The steps include: (1) fresh spur roots and fresh grape roots are respectively added with water, and the mixture is initially crushed and then solid-liquid separated to obtain liquid I and solid; the solid is dried and then crushed step by step to powders of 800-1500 mesh; liquid I is solid-liquid separated to obtain liquid II and a slurry; and finally the powder and the slurry are mixed to obtain a mixture; (2) Adding borneol water or natural borneol to the mixture of step (1), mixing thoroughly to obtain the original drug intermediate, and refrigerating for later use.

10. The preparation method according to claim 9, characterized in that: The steps of crushing the spur root and the jade grape step by step are: Fresh spur roots or jade grape roots are respectively added with water, first crushed to 80-100 mesh, and the solid-liquid separation is performed to obtain liquid I and solid; after the solid is dried, it is crushed step by step according to 200 mesh and 800 mesh, and finally crushed to powder of 800-1500 mesh; Liquid I is solid-liquid separated to obtain liquid II and slurry, wherein the volume of liquid II is 60%-80% of the volume of liquid I; finally, the powder is mixed with the slurry to obtain a mixture.

11. Use of the Chinese medicinal composition for treating burns and scalds according to any one of claims 1 to 8 in the preparation of medicines for treating burns and scalds.

12. The use according to claim 11, characterized in that: The burn and scald diseases include wound infection, body fluid exudation, edema and pain symptoms caused by burns and scalds.

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