External composition and application thereof

Abstract

The invention discloses an external composition which contains a traditional Chinese medicine composition, and the traditional Chinese medicine composition comprises fructus cnidii, radix sophorae flavescentis and cortex dictamni. The external composition can be applied to preparation of a regulating agent for regulating and controlling the reduction of the concentration of neuropeptide and / or histamine in blood. The external composition can act on a skin target area where pruritus occurs, the concentration of P substances, gastrin releasing peptide and histamine in blood can be regulated and controlled, and then the attack of symptoms such as pruritus and the like can be relieved or treated.

Description

Technical Field

[0001] The present invention relates to the technical field of external regulating agents, and in particular to an external composition and application thereof. Background Art

[0002] Pruritus is a common skin disease characterized by itching. It is a typical clinical symptom, primarily manifested by erythema, papules, and unbearable itching. Chronic cases present with infiltration and hypertrophy, making complete cure difficult and highly recurrent. The skin of pruritus patients is inherently fragile, and scratching can damage the skin's integrity, increase sebum secretion in lesions, and allow normal surface bacteria to proliferate, becoming opportunistic pathogens and impairing skin repair.

[0003] Substance P (SP) is a neuropeptide and an important substance that mediates itching. When the concentration of substance P in the body's blood is abnormally high, it will trigger an itching attack. If the concentration of substance P in the blood is reduced through regulators or drug regulation, it will help patients with pruritus relieve itching symptoms and other discomfort symptoms.

[0004] Gastrin-releasing peptide (GRP) is a neuropeptide that is involved in a wide range of physiological functions, including exocrine and endocrine functions, smooth muscle contraction, pain and itch transmission, satiety, etc. Gastrin-releasing peptide (GRP) is also involved in the pathogenesis of atopic dermatitis. When the concentration of gastrin-releasing peptide (GRP) in the body's blood is abnormally elevated, it will trigger an itching attack. If the concentration of gastrin-releasing peptide (GRP) in the blood is reduced through regulators or drug regulation, it will help patients with pruritus relieve itching symptoms and other discomfort symptoms.

[0005] Histamine is a chemical mediator secreted by basophils and mast cells. It is found in higher concentrations in the skin, bronchial mucosa, intestinal mucosa and nervous system. Its receptors are members of the G protein-coupled receptor family, of which histamine receptor 1 and histamine receptor 4 play an important role in the occurrence of pruritus. When the body is subjected to various stimuli or an allergic reaction occurs, histamine is released and binds to histamine receptors to produce biological effects. Histamine is one of the important mediators involved in the pathological and physiological processes of pruritus and is an important source of pruritus. When the concentration of histamine in the body's blood is abnormally elevated, it can trigger an itch attack. If the concentration of histamine in the blood is reduced through regulators or drug regulation, it will help patients with pruritus to relieve symptoms of pruritus and other discomfort symptoms.

[0006] If a regulator is developed to reduce the concentration of the corresponding indicator, such as an abnormal increase in the concentration of substance P in the body's blood, or an abnormal increase in the concentration of gastrin-releasing peptide (GRP), or an abnormal increase in the concentration of histamine, the itching attack caused by the corresponding increase in the concentration of the corresponding indicator will be alleviated or treated. Summary of the Invention

[0007] The present invention aims to provide a topical composition and its use, wherein the topical composition of the present invention can be used in the preparation of a regulating agent for regulating the reduction of neuropeptide and / or histamine concentrations in the blood. The topical composition of the present invention can be applied to a target area of skin where itching occurs (e.g., by spraying, smearing, or coating), thereby regulating the reduction of blood concentrations of substance P, gastrin-releasing peptide (GRP), and histamine, thereby alleviating or treating symptoms such as itching, urticaria, eczema, and atopic dermatitis. The topical composition can also be used as a skin repair agent to repair skin damage / skin discomfort symptoms of the corresponding symptoms.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0009] A composition for external use, comprising a traditional Chinese medicine composition comprising Cnidium monnieri, Sophora flavescens, and Dictamnus chinensis root;

[0010] The preparation method of the Chinese medicine composition comprises the following steps:

[0011] Step (1): grinding the components of the traditional Chinese medicine composition (Cnidium monnieri, Sophora flavescens, and Dictamnus cortex) separately and mixing them to prepare a mixture;

[0012] Step (2): adding ethanol to the mixture obtained in step (1), soaking and extracting, filtering and collecting the filtrate, wherein the filtrate is a Chinese medicine extract, and the Chinese medicine composition is obtained;

[0013] The external composition is used in preparing a regulator for regulating the reduction of neuropeptide and / or histamine concentration in blood.

[0014] That is, when the present invention is put into practical use, the topical composition of the present invention can be used as a regulator for preparing a control agent for regulating the reduction of neuropeptide concentration in the blood; or, the topical composition of the present invention can be used as a regulator for preparing a control agent for regulating the reduction of histamine concentration in the blood; or, the topical composition of the present invention can be used as a regulator for preparing a control agent for simultaneously regulating the reduction of neuropeptide and histamine concentrations in the blood.

[0015] Preferably, when the external-use composition of the present invention is used as a regulator for preparing a regulator for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide includes substance P (SP) and / or gastrin-releasing peptide (GRP).

[0016] That is, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be substance P (Substances P, SP); or, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be gastrin-releasing peptide (GRP); or, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be substance P (Substances P, SP) and gastrin-releasing peptide (GRP).

[0017] When the topical composition of the present invention is used as a regulator for preparing a regulator for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide is a class of biologically active peptide substances secreted by neurons, including substance P (SP), gastrin-releasing peptide (GRP), etc. The neuropeptide plays a key role in the process of inducing itching.

[0018] Substance P (SP) is an 11-amino acid neuropeptide primarily secreted by neurons but also expressed in a variety of non-neuronal cells, such as microglia, epithelial cells, stem cells, and immune cells, exerting its effects in both autocrine and paracrine ways. SP can regulate various inflammatory and skin-related itching processes, producing symptoms such as rashes, urticaria, and itching, as seen in skin diseases such as eczema, urticaria, psoriasis, and asthma.

[0019] When the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of the concentration of neuropeptide substance P (Substances P, SP) in the blood: when the topical composition of the present invention takes effect, it can inhibit the transmission of pain from peripheral nerves to the central nervous system by regulating the reduction of the concentration of neuropeptide substance P in the blood, thereby achieving local analgesia. It can be used in arthritis, muscle strain, postoperative incision pain, neuralgia and other scenes requiring local analgesia, can reduce the itch-scratch cycle, and can be used to relieve itching, erythema and skin barrier repair, etc.

[0020] The gastrin-releasing peptide receptor (GRPR), a pruritus-specific receptor in the dorsal horn of the spinal cord, mediates a specific itch signaling pathway. Gastrin-releasing peptide (GRP), a 27-amino acid peptide, is a ligand for GRPR. GRP binds to and is internalized by GRPR neurons, inducing itch. Peripheral blood GRP levels are positively correlated with the severity and itch of atopic dermatitis.

[0021] When the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of the concentration of the neuropeptide gastrin-releasing peptide (GRP) in the blood: when the topical composition of the present invention takes effect, it can regulate the reduction of the concentration of the neuropeptide gastrin-releasing peptide in the blood, inhibit the local release of GRP, block the cascade reaction of inflammatory cytokines, and thus relieve the symptoms of erythema, scaling and itching. Chronic wounds (such as diabetic foot ulcers) are often accompanied by persistent inflammation and elevated levels of GRP. The regulator of the present invention can inhibit GRP-mediated neurogenic inflammation, reduce the excessive release of proteases and free radicals by macrophages, promote granulation tissue formation and epidermal regeneration. The regulator of the present invention can inhibit the release of local GRP, reduce the concentration of GRP, relieve neurodermatitis, itching, reduce neuroinflammation and hyperalgesia, etc.

[0022] Histamine is a chemical mediator secreted by basophils and mast cells. It is found in high concentrations in the skin, bronchial mucosa, intestinal mucosa, and nervous system. Its receptors are members of the G protein-coupled receptor family, with histamine receptor 1 and histamine receptor 4 playing a key role in the development of pruritus. When the body is exposed to various physical and chemical stimuli or an allergic reaction, histamine is released, binding to histamine receptors and producing biological effects. Histamine is a key mediator involved in the pathological and physiological processes of pruritus and is a major itch-causing factor.

[0023] When the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of histamine concentration in the blood: when the topical composition of the present invention takes effect, it can directly inhibit mast cell degranulation and histamine release by regulating the reduction of histamine concentration in the blood, block H1 receptor-mediated vasodilation, itching and edema, and relieve acute allergic reactions (such as urticaria, contact dermatitis); it can be sprayed or applied through the nasal mucosa or the skin around the eyes to inhibit local histamine release, reduce nasal congestion, tearing and sneezing; it can inhibit histamine-induced Th2 type immune response, break the vicious cycle of "itch-scratching-inflammation", and relieve the symptoms of atopic dermatitis (eczema); it can reduce the peak histamine level in the blood, reduce increased vascular permeability and recurrence of wheals, and achieve the relief of chronic urticaria symptoms; it can inhibit histamine release at the postoperative incision site, reduce the risk of scar hyperplasia, etc.

[0024] The topical composition of the present invention can be applied to the target area of the skin where itching occurs (e.g., by spraying, smearing, coating, etc.), and can regulate the reduction of the concentrations of substance P, gastrin-releasing peptide (GRP), and histamine in the blood, thereby relieving or treating symptoms such as itching, urticaria, eczema, and allergic dermatitis, and can also repair the skin according to the corresponding skin damage / skin discomfort symptoms.

[0025] Preferably, in parts by weight, the weight portions of the components in the traditional Chinese medicine composition are: 30 parts of Cnidium monnieri, 20 parts of Sophora flavescens, and 15 parts of Dictamnus cortex.

[0026] Preferably, in step (1), each component in the traditional Chinese medicine composition is pulverized separately and then passed through a 20-mesh sieve.

[0027] Preferably, in step (2), 8 times the amount of ethanol is added to the mixture obtained in step (1) by weight (that is, 8 parts by weight of ethanol are added to 1 part by weight of the mixture obtained in step (1)), the volume percentage of the ethanol is 50%, and the soaking time is 7 days.

[0028] Preferably, the external-use composition further contains a biological agent; the biological agent comprises a fermentation product of Bacillus velezensis and / or its metabolites.

[0029] That is, the biological preparation contains the fermentation product of Bacillus Velez; or, the biological preparation contains the metabolite of Bacillus Velez; or, the biological preparation contains the fermentation product of Bacillus Velez and the metabolite of Bacillus Velez.

[0030] In the present invention, the Bacillus Velez fermented product is a composition in which Bacillus Velez is grown and propagated in a culture medium. The Bacillus Velez fermented product is a cell culture supernatant of Bacillus Velez, from which cells have been removed. Specifically, the fermented broth is centrifuged and then sterilized by filtration to remove cells. Furthermore, the fermented product (cell culture supernatant) is obtained by filtering the culture medium in which the Bacillus Velez cells were cultured.

[0031] In the present invention, the metabolites of Bacillus velez include but are not limited to primary metabolites, soluble metabolites, fatty acids, sugars, sugar alcohols, peptides, proteins, nucleotides, secondary metabolites, polynucleotides and polysaccharides of Bacillus velez.

[0032] Preferably, the method for preparing the biological agent comprises the following steps:

[0033] Bacillus velezensis was inoculated into a liquid culture medium at an inoculum rate of 0.1%, and cultured at 37°C for 48 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 5000 r / min for 5 minutes, and then filtered through a 0.22 μm filter membrane to remove bacteria to obtain the biological preparation.

[0034] Preferably, the Bacillus velezensis is Bacillus velezensis EYWS-B01, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025481.

[0035] The Bacillus velezensis EYWS-B01 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on March 17, 2025, with the deposit number being CCTCC NO: M2025481.

[0036] Preferably, in the external-use composition, the weight ratio of the traditional Chinese medicine composition to the biological agent is 6:4, and the external-use composition is obtained by uniformly mixing the two.

[0037] Preferably, the liquid culture medium comprises the following components by weight: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, and 96% distilled water. The liquid culture medium is a Bacillus liquid culture medium.

[0038] Preferably, the preparation method of the liquid culture medium is: weighing each formula component of the liquid culture medium according to weight percentage, mixing each formula component to prepare a mixed solution, sterilizing the mixed solution at 121° C. for 20 minutes, and naturally cooling to room temperature to prepare the liquid culture medium.

[0039] Preferably, the topical composition is used in the preparation of a medicament for relieving or treating itching, urticaria, eczema, allergic dermatitis, or skin repair.

[0040] The topical composition of the present invention can be used as a regulator for regulating the reduction of neuropeptide and / or histamine concentrations in the blood. When the topical composition is put into practical use, the topical composition can be specifically used as a drug for relieving or treating itching, urticaria, eczema, allergic dermatitis, and skin repair.

[0041] The drug of the present invention prepared by the external use composition can be applied to the target skin area where itching occurs (such as by spraying, smearing, coating, etc.), and can regulate the reduction of the concentration of substance P, gastrin-releasing peptide (GRP), and histamine in the blood, thereby relieving or treating symptoms such as itching, urticaria, eczema, and allergic dermatitis. It can also repair the skin according to the skin damage / skin discomfort symptoms of the corresponding symptoms.

[0042] Preferably, the drug is any one of a spray, lotion, gel, cream, emulsion, paste, and ointment.

[0043] Preferably, the medicine is a spray.

[0044] When the drug is used in the form of a spray, the spray is more convenient to use, can achieve rapid penetration, and can be used for symptoms such as acute itching.

[0045] Preferably, the method for applying the drug is as follows: the drug is put into a spray bottle.

[0046] The drug is put into a spray bottle, and the spray bottle can be pressed to squeeze out an appropriate amount of the drug to be smeared or applied to the target area of the skin. Alternatively, an appropriate amount of the drug can be directly sprayed on the target area of the skin, which is more flexible and convenient to use.

[0047] Beneficial effects of the present invention: The topical composition of the present invention can be used in the preparation of a regulator for regulating the reduction of neuropeptide and / or histamine concentrations in the blood. The topical composition of the present invention can be used as a regulator to act on the target skin area where itching occurs (e.g., by spraying, smearing, coating, etc.), thereby regulating the reduction of substance P, gastrin-releasing peptide (GRP), and histamine concentrations in the blood, thereby relieving or treating symptoms such as itching, urticaria, eczema, and atopic dermatitis. It can also repair skin damage / skin discomfort symptoms caused by corresponding symptoms and can be used as a skin repair agent. The topical composition of the present invention can also inhibit a variety of pathogenic bacteria. By applying the topical composition of the present invention, the purposes of antibacterial and antipruritic can be achieved. DETAILED DESCRIPTION

[0048] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below.

[0049] Example 1:

[0050] Screening and identification of strains

[0051] Use sterile sample bottles to take the well water on Yaowang Mountain in Feihuling, Liulin Township, Huangmei County, Huanggang City, Hubei Province, dilute the well water 10 times, and take 10 -3 , 10 -4 , 10-5 100 μL of the sample dilutions of three dilutions were spread on nutrient agar medium and cultured in a 37°C incubator for 48 h. Single colonies were picked and repeatedly isolated and purified using the partitioning and streaking method, and the morphological characteristics of each colony were recorded.

[0052] The purified strains were streaked on nutrient agar medium and cultured in a 37°C incubator for 24 h. Single colonies were picked and inoculated into 50 mL of nutrient broth. After culture at 37°C and 200 r / min for 24 h, seed liquid was obtained. The seed liquid was inoculated into 100 mL of nutrient broth at a ratio of 1%, and cultured at 37°C for 48 h to obtain fermentation liquid. The fermentation liquid was centrifuged at 5000 r / min for 5 min to obtain the supernatant, which was then filtered through a 0.22 μm sterile filter membrane for later use.

[0053] Streak E. coli, Staphylococcus aureus, Candida albicans, Trichophyton rubrum, and Epidermophyton floccosum on a plate and incubate at 36°C for 20 hours. Pick single colonies of E. coli and Staphylococcus aureus and inoculate them into nutrient broth. Pick single colonies of Candida albicans, Trichophyton rubrum, and Epidermophyton floccosum and inoculate them into PDA liquid medium. Incubate at 37°C with shaking for 16 hours. Adjust the cultured bacterial suspension to a bacterial count of 1×10 7 cfu / mL. Take 100μL of indicator bacterial suspension and drop it on nutrient agar (Escherichia coli, Staphylococcus aureus) or PDA (Candida albicans, Trichophyton rubrum, Epidermophyton floccosum) plate, spread it evenly with a spreading stick, use sterile tweezers to take the sterilized Oxford cup, and gently place it on the surface of the nutrient agar medium. Use a micropipette to draw 200μL of the prepared bacterial supernatant and inject it into the Oxford cup placed steadily. Place the culture medium in a 4℃ refrigerator for 8h, then place the plate after the diffusion treatment in a constant temperature incubator at 37℃. After culturing for 16h-24h, record the antibacterial test results of the target strain. See Table 1 for details.

[0054] Table 1 Antibacterial test results of target strains

[0055]

[0056] From the antibacterial test results of the target strains in Table 1, it can be seen that the target strains screened in the present invention have good antibacterial effects on pathogenic bacteria Escherichia coli, Staphylococcus aureus, Candida albicans, Trichophyton rubrum, and Epidermophyton floccosum.

[0057] The target strain obtained by screening was sent to Aoke (Wuhan) Biotechnology Co., Ltd. for gene sequencing. The comparison results of the NCBI database showed that the isolated strain had a 100% homology with sequences such as Bacillus velezensis strain BIM B-454D, Bacillus velezensis strain SRCM103691, and Bacillus velezensis strain AB191. The 16s rRNA sequence of the strain is shown in SEQ NO.1. The strain belongs to Bacillus velezensis and is classified as Bacillus velezensis EYWS-B01; Bacillus velezensis EYWS - B01.

[0058] SEQ NO.1

[0059]

[0060] Bacillus Velez is a beneficial bacterium that can produce a variety of bioactive substances and has good biological functions.

[0061] Example 2:

[0062] A composition for external use contains a traditional Chinese medicine composition comprising Cnidium monnieri, Sophora flavescens and Dictamnus cortex.

[0063] The preparation method of the Chinese medicine composition comprises the following steps:

[0064] Step (1): grinding each component of the Chinese medicine composition (in parts by weight, the weight parts of each component in the Chinese medicine composition are: 30 parts of Cnidium monnieri, 20 parts of Sophora flavescens, and 15 parts of Dictamnus cortex) respectively, and mixing them to prepare a mixture;

[0065] Step (2): adding ethanol to the mixture obtained in step (1), soaking and extracting, filtering through 6 layers of gauze to collect the filtrate, wherein the filtrate is a Chinese medicine extract, and the Chinese medicine composition, i.e., the external use composition of the present invention, is obtained.

[0066] The prepared external use composition can be put into a spray bottle, and the spray bottle can be pressed to squeeze out an appropriate amount of the external use composition and smeared or applied to the target area of the skin. Alternatively, an appropriate amount of the external use composition can be directly sprayed on the target area of the skin, which is more flexible and convenient to use.

[0067] The use of the topical composition in the preparation of a regulator for regulating the reduction of neuropeptide and / or histamine concentrations in the blood, that is, when the topical composition of the present invention is actually used, the topical composition of the present invention can be used as a regulator for regulating the reduction of neuropeptide concentrations in the blood as needed; or, as needed, the topical composition of the present invention can also be used as a regulator for regulating the reduction of histamine concentrations in the blood; or, as needed, the topical composition of the present invention can also be used as a regulator for regulating the simultaneous reduction of neuropeptide and histamine concentrations in the blood.

[0068] When the external-use composition of the present invention is used as described above, the neuropeptide includes substance P (SP) and / or gastrin-releasing peptide (GRP).

[0069] That is, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be substance P (Substances P, SP); or, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be gastrin-releasing peptide (GRP); or, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be substance P (Substances P, SP) and gastrin-releasing peptide (GRP).

[0070] The topical composition of the present invention can be applied to the target area of the skin where itching occurs (such as by spraying, smearing, coating, etc.), and can regulate the reduction of the concentration of substance P, gastrin-releasing peptide (GRP), and histamine in the blood, thereby relieving or treating symptoms such as itching, urticaria, eczema, and allergic dermatitis. It can also repair the skin according to the skin damage / skin discomfort symptoms of the corresponding symptoms.

[0071] In parts by weight, the weight portions of the components in the traditional Chinese medicine composition are: 30 parts of Cnidium monnieri, 20 parts of Sophora flavescens, and 15 parts of Dictamnus cortex.

[0072] In the step (1), each component in the traditional Chinese medicine composition is crushed separately and then passed through a 20-mesh sieve.

[0073] In the step (2), 8 times the amount of ethanol is added to the mixture obtained in the step (1) by weight (i.e., 8 parts by weight of ethanol are added to 1 part by weight of the mixture obtained in the step (1)), the volume percentage of the ethanol is 50%, and the soaking time is 7 days.

[0074] The external-use composition can be used in the preparation of medicines for relieving or treating itching, urticaria, eczema, allergic dermatitis, and skin repair, and the external-use composition can be used as the main component of the corresponding medicines.

[0075] The medicine can be any one of a spray, a lotion, a gel, a cream, an emulsion, a paste, and an ointment.

[0076] The topical composition can be used as the main component of the corresponding drug, and pharmaceutically acceptable excipients or auxiliary ingredients can be added to prepare a pharmaceutical preparation commonly used in pharmacy. Furthermore, according to the needs of use, the drug can be made into various dosage forms, for example, the drug can be made into a spray, lotion, gel, cream, emulsion, paste, ointment, etc., and any one of these dosage forms can be selected according to needs.

[0077] The drug of the present invention prepared by the external use composition can be applied to the target skin area where itching occurs (such as by spraying, smearing, coating, etc.), and can regulate the reduction of the concentration of substance P, gastrin-releasing peptide (GRP), and histamine in the blood, thereby relieving or treating symptoms such as itching, urticaria, eczema, and allergic dermatitis. It can also repair the skin according to the skin damage / skin discomfort symptoms of the corresponding symptoms.

[0078] Preferably, the drug is in the form of a spray. When the drug is used in the form of a spray, the spray is more convenient to use, can achieve rapid penetration, and can be used for symptoms such as acute itching.

[0079] Method of application of the drug: put the drug into a spray bottle.

[0080] The drug is put into a spray bottle, and the spray bottle can be pressed to squeeze out an appropriate amount of the drug to be smeared or applied to the target area of the skin. Alternatively, an appropriate amount of the drug can be directly sprayed on the target area of the skin, which is more flexible and convenient to use.

[0081] Example 3:

[0082] The method for preparing the biological agent of the present invention comprises the following steps:

[0083] Bacillus velezensis was inoculated into a liquid culture medium at an inoculum rate of 0.1%, and cultured at 37°C for 48 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 5000 r / min for 5 minutes, and then filtered through a 0.22 μm filter membrane to remove bacteria to obtain the biological preparation.

[0084] The Bacillus velezensis is Bacillus velezensis EYWS-B01, that is, the above classification is named Bacillus velezensis EYWS-B01; Bacillus velezensis EYWS - The strain B01 is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025481.

[0085] The Bacillus velezensis EYWS-B01 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on March 17, 2025, with the deposit number being CCTCC NO: M2025481.

[0086] The liquid culture medium comprises the following components by weight: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, and 96% distilled water. The liquid culture medium is a Bacillus liquid culture medium.

[0087] The preparation method of the liquid culture medium comprises: weighing the respective formula components of the liquid culture medium according to weight percentage, mixing the respective formula components to prepare a mixed liquid, sterilizing the mixed liquid at 121° C. for 20 minutes, and naturally cooling to room temperature to prepare the liquid culture medium.

[0088] Example 4:

[0089] A composition for external use, comprising a traditional Chinese medicine composition and a biological agent; in the composition for external use, the weight ratio of the traditional Chinese medicine composition to the biological agent is 6:4, and the composition for external use is obtained by uniformly mixing the traditional Chinese medicine composition and the biological agent.

[0090] The biological preparation comprises a fermentation product of Bacillus velez and / or a metabolite thereof.

[0091] The prepared external use composition can be put into a spray bottle, and the spray bottle can be pressed to squeeze out an appropriate amount of the external use composition and smeared or applied to the target area of the skin. Alternatively, an appropriate amount of the external use composition can be directly sprayed on the target area of the skin, which is more flexible and convenient to use.

[0092] The traditional Chinese medicine composition comprises Cnidium monnieri, Sophora flavescens and Dictamnus cortex.

[0093] The preparation method of the Chinese medicine composition comprises the following steps:

[0094] Step (1): grinding each component of the Chinese medicine composition (in parts by weight, the weight parts of each component in the Chinese medicine composition are: 30 parts of Cnidium monnieri, 20 parts of Sophora flavescens, and 15 parts of Dictamnus cortex) respectively, and mixing them to prepare a mixture;

[0095] Step (2): adding ethanol to the mixture obtained in step (1), soaking and extracting, filtering through 6 layers of gauze to collect the filtrate, wherein the filtrate is a Chinese medicine extract, and the Chinese medicine composition is obtained.

[0096] The use of the topical composition in the preparation of a regulator for regulating the reduction of neuropeptide and / or histamine concentrations in the blood, that is, when the topical composition of the present invention is actually used, the topical composition of the present invention can be used as a regulator for regulating the reduction of neuropeptide concentrations in the blood as needed; or, as needed, the topical composition of the present invention can also be used as a regulator for regulating the reduction of histamine concentrations in the blood; or, as needed, the topical composition of the present invention can also be used as a regulator for regulating the simultaneous reduction of neuropeptide and histamine concentrations in the blood.

[0097] When the external-use composition of the present invention is used as described above, the neuropeptide includes substance P (SP) and / or gastrin-releasing peptide (GRP).

[0098] That is, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be substance P (Substances P, SP); or, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be gastrin-releasing peptide (GRP); or, when the topical composition of the present invention is used as a regulator for preparing a control agent for regulating the reduction of neuropeptide and / or histamine concentration in the blood, the neuropeptide may be substance P (Substances P, SP) and gastrin-releasing peptide (GRP).

[0099] The topical composition of the present invention can be applied to the target area of the skin where itching occurs (such as by spraying, smearing, coating, etc.), and can regulate the reduction of the concentration of substance P, gastrin-releasing peptide (GRP), and histamine in the blood, thereby relieving or treating symptoms such as itching, urticaria, eczema, and allergic dermatitis. It can also repair the skin according to the skin damage / skin discomfort symptoms of the corresponding symptoms.

[0100] In parts by weight, the weight portions of the components in the traditional Chinese medicine composition are: 30 parts of Cnidium monnieri, 20 parts of Sophora flavescens, and 15 parts of Dictamnus cortex.

[0101] In the step (1), each component in the traditional Chinese medicine composition is crushed separately and then passed through a 20-mesh sieve.

[0102] In the step (2), 8 times the amount of ethanol is added to the mixture obtained in the step (1) by weight (i.e., 8 parts by weight of ethanol are added to 1 part by weight of the mixture obtained in the step (1)), the volume percentage of the ethanol is 50%, and the soaking time is 7 days.

[0103] In this Example 4, the preparation method of the above-mentioned traditional Chinese medicine composition is the same as the preparation method of the traditional Chinese medicine composition in Example 2.

[0104] In this embodiment 4, the method for preparing the biological agent comprises the following steps:

[0105] Bacillus velezensis was inoculated into a liquid culture medium at an inoculum rate of 0.1%, and cultured at 37°C for 48 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 5000 r / min for 5 minutes, and then filtered through a 0.22 μm filter membrane to remove bacteria to obtain the biological preparation.

[0106] The Bacillus velezensis is Bacillus velezensis EYWS-B01, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2025481.

[0107] The Bacillus velezensis EYWS-B01 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on March 17, 2025, with the deposit number being CCTCC NO: M2025481.

[0108] The liquid culture medium comprises the following components by weight: 1% peptone, 1.5% beef extract, 1.5% sodium chloride, and 96% distilled water. The liquid culture medium is a Bacillus liquid culture medium.

[0109] The preparation method of the liquid culture medium comprises: weighing the respective formula components of the liquid culture medium according to weight percentage, mixing the respective formula components to prepare a mixed liquid, sterilizing the mixed liquid at 121° C. for 20 minutes, and naturally cooling to room temperature to prepare the liquid culture medium.

[0110] In this Example 4, the preparation method of the above-mentioned biological preparation is the same as the preparation method of the biological preparation in Example 3.

[0111] The external composition is used in preparing medicines for relieving or treating itching, urticaria, eczema, allergic dermatitis and skin repair.

[0112] The topical composition of the present invention can be used as a regulator for regulating the reduction of neuropeptide and / or histamine concentrations in the blood. When the topical composition is put into practical use, the topical composition can be specifically used as a drug for relieving or treating itching, urticaria, eczema, allergic dermatitis, and skin repair.

[0113] The drug of the present invention prepared by the external use composition can be applied to the target skin area where itching occurs (such as by spraying, smearing, coating, etc.), and can regulate the reduction of the concentration of substance P, gastrin-releasing peptide (GRP), and histamine in the blood, thereby relieving or treating symptoms such as itching, urticaria, eczema, and allergic dermatitis, and can also repair the skin according to the corresponding skin damage / skin discomfort symptoms.

[0114] The medicine can be any one of a spray, a lotion, a gel, a cream, an emulsion, a paste, and an ointment.

[0115] The topical composition can be used as the main component of the corresponding drug, and pharmaceutically acceptable excipients or auxiliary ingredients can be added to prepare a pharmaceutical preparation commonly used in pharmacy. Furthermore, according to the needs of use, the drug can be made into various dosage forms, for example, the drug can be made into a spray, lotion, gel, cream, emulsion, paste, ointment, etc., and any one of these dosage forms can be selected according to needs.

[0116] Preferably, the drug is in the form of a spray. When the drug is used in the form of a spray, the spray is more convenient to use, can achieve rapid penetration, and can be used for symptoms such as acute itching.

[0117] Method of application of the drug: put the drug into a spray bottle.

[0118] The drug is put into a spray bottle, and the spray bottle can be pressed to squeeze out an appropriate amount of the drug to be smeared or applied to the target area of the skin. Alternatively, an appropriate amount of the drug can be directly sprayed on the target area of the skin, which is more flexible and convenient to use.

[0119] Test example:

[0120] Animal model establishment:

[0121] Forty-eight healthy mice, half male and half female, were selected. One day before the experiment, the hair on the back of each mouse was shaved, covering an area of approximately 2 cm × 2 cm. The mice were randomly divided into six groups according to sex and weight, with 8 mice in each group, namely blank control group, model group, positive control group, test group 1, test group 2, and test group 3. The test substance A was evenly sprayed on the shaved area (test area) on the back of each group of mice, with the spraying area of approximately 1.5 cm × 1.5 cm, of which:

[0122] The blank control group was sprayed with test substance A, which was normal saline, 50 μL / time, once / day, for 7 consecutive days;

[0123] The model group, positive control group, test group 1, test group 2 and test group 3 were all sprayed with test substance A, which was 2,4-dinitrochloropropiophenone solution (1%), 50 μL / time, once / day, for 7 consecutive days.

[0124] The specific processing methods are as follows:

[0125] Blank control group: Test substance A was sprayed with normal saline, 50 μL / time, once / day, for 7 consecutive days;

[0126] Model group: The test substance A administered by spraying was 2,4-dinitrochloropropiophenone solution (1%), 50 μL / time, once / day, for 7 consecutive days;

[0127] Positive control group: Test substance A was sprayed with 2,4-dinitrochloropropiophenone solution (1%), 50 μL / time, once / day, for 7 consecutive days;

[0128] Test group 1: The test substance A administered by spraying was 2,4-dinitrochloropropiophenone solution (1%), 50 μL / time, once / day, for 7 consecutive days;

[0129] Test group 2: The test substance A administered by spraying was 2,4-dinitrochloropropiophenone solution (1%), 50 μL / time, once / day, for 7 consecutive days;

[0130] Test group 3: Test substance A administered by spraying was 2,4-dinitrochloropropiophenone solution (1%), 50 μL / time, once / day, for 7 consecutive days.

[0131] At the same time, 4 hours after the spraying of the test substance A (2,4-dinitrochlorophenylpropiophenone solution (1%)) was completed, the five groups were sprayed with the test substance B on the hair removal area, as follows:

[0132] Model group: Test substance B administered by spraying was normal saline.

[0133] Positive control group: Test substance B administered by spraying was terbinafine hydrochloride spray.

[0134] Test Group 1: The test substance B administered by spraying was the external use composition prepared in Example 2 of the present application.

[0135] Test Group 2: The test substance B administered by spraying was the biological preparation prepared in Example 3 of the present application.

[0136] Test group 3: The test substance B administered by spraying was the external use composition prepared in Example 4 of the present application.

[0137] After the test substance B was sprayed on the five groups, they all pressed the hair removal area downward continuously for three times starting from 5 cm above the hair removal area sprayed with the test substance B.

[0138] Behavioral observation: Every day, 20 minutes after the spraying of test substance B, the number of pruritus attacks in each group of mice within 10 minutes was recorded with a video camera, and the pruritus inhibition rate was calculated. The statistical results of the number of pruritus attacks (scratching times) and pruritus inhibition rate of each group of mice are shown in Table 2.

[0139] The calculation method of pruritus inhibition rate is as follows:

[0140] Itch inhibition rate of the positive control group = (number of scratches in the model group - number of scratches in the positive control group) ÷ number of scratches in the model group × 100%.

[0141] Itch inhibition rate of test group 1 = (number of scratches in the model group - number of scratches in test group 1) ÷ number of scratches in the model group × 100%.

[0142] Itch inhibition rate of test group 2 = (number of scratches in the model group - number of scratches in test group 2) ÷ number of scratches in the model group × 100%.

[0143] Itch inhibition rate of test group 3 = (number of scratches in the model group - number of scratches in test group 3) ÷ number of scratches in the model group × 100%.

[0144] Symptoms of pruritus in mice include: scratching the head with the front paws, scratching the trunk with the hind paws, and biting various parts of the body or the tail with the mouth.

[0145] If the mice in each group experienced any of the above-mentioned symptoms of pruritus, it was recorded as one pruritus attack (also recorded as one scratching frequency in Table 2).

[0146] At the same time, the sensory status of the skin of each group of mice was observed and recorded throughout the whole process, including whether there were erythema, edema, and lichenification.

[0147] Serum cytokine assays: 20 minutes after the final spraying of test substance B, mice were anesthetized with sodium pentobarbital, and blood samples were collected to obtain serum. Finally, the mice were sacrificed. Serum concentrations of substance P (SP), gastrin-releasing peptide (GRP), and histamine were measured using enzyme-linked immunosorbent assay kits (Wuhan Cloud-Clone Technology Co., Ltd.). The corresponding test results are shown in Table 3.

[0148] Table 2 Statistical results of the number of pruritus attacks in mice

[0149] Group Number of scratching (times / day) Itch inhibition rate (%) Blank control group 0.68±0.24 Model Group 10.95±2.39 Positive control group <![CDATA[2.36±0.24 *** ]]> 78.45 Experimental group 1 <![CDATA[5.11±0.37 *** ]]> 53.33 Experimental Group 2 10.41±0.33 4.93 Experimental Group 3 <![CDATA[2.27±0.28 *** ]]> 79.27

[0150] Note: Compared with the model group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001; no significant difference was performed on the blank group, and there was no significant difference between the experimental group 2 and the model group (p > 0.05).

[0151] Compared with the blank control group, the scratching frequency of mice in the model group was significantly increased.

[0152] The skin sensory status of mice in each group is as follows:

[0153] Blank control group: There was no change in the skin sensory state of the mouse shaved area (test area).

[0154] Model group: After the above-mentioned spraying of test substance A, 2,4-dinitrochloropropiophenone solution (1%), the skin sensory organs of the shaved area (test area) of the mice showed erythema, edema, and lichenification.

[0155] Positive control group: After the above-mentioned spraying and administration of the test substance A, which was 2,4-dinitrochlorophenylpropiophenone solution (1%), the skin sensory manifestations of the shaved area (test area) of the mice showed erythema, edema, and lichenification. Furthermore, after the above-mentioned spraying and administration of the test substance B, which was terbinafine hydrochloride spray, the erythema, edema, and lichenification sensory manifestations of the skin sensory manifestations of the shaved area (test area) of the mice were alleviated.

[0156] Test group 1: After the above-mentioned spraying and administration of the test substance A, which was a 2,4-dinitrochlorophenylpropiophenone solution (1%), the skin sensory organs of the mouse's hair-cut area (test area) showed erythema, edema, and lichenification. Furthermore, thereafter, after the above-mentioned spraying and administration of the test substance B, which was the external use composition prepared by Example 2 of the present application, the erythema, edema, and lichenification sensory organs of the mouse's hair-cut area (test area) were significantly alleviated.

[0157] Test Group 2: After the above-mentioned spraying and administration of the test substance A, which was a 2,4-dinitrochlorophenylpropiophenone solution (1%), the skin sensory organs of the mouse's hair-removed area (test area) showed erythema, edema, and lichenification. Furthermore, after the above-mentioned spraying and administration of the test substance B, which was the biological preparation prepared by Example 3 of the present application, the sensory organs of the mouse's hair-removed area (test area) showed no significant relief of the erythema, edema, and lichenification.

[0158] Test group 3: After the above-mentioned spraying and administration of the test substance A, which was a 2,4-dinitrochlorophenylpropiophenone solution (1%), the skin sensory organs of the mouse's hair removal area (test area) showed erythema, edema, and lichenification. Furthermore, thereafter, after the above-mentioned spraying and administration of the test substance B, which was the external use composition prepared by Example 4 of the present application, the erythema, edema, and lichenification sensory organs of the mouse's hair removal area (test area) were significantly alleviated, and the degree of relief was better than that of the positive control group and test group 1.

[0159] By observing the sensory state of mouse skin and comparing it with the model group, it was found that the positive control group, experimental group 1 and experimental group 3 could improve the skin condition of mice. The skin erythema, edema, epidermal exfoliation and lichenification of the mice's hair-cut area (experimental area) were improved, and the degree of itching was relieved.

[0160] As can be seen from Table 2, there was no significant change in the number of scratchings between the test group 2 and the model group (p>0.05), indicating that the biological preparation prepared by Example 3 of the present application had no effect on alleviating itching symptoms.

[0161] Compared with the model group, the number of scratching of mice in test group 1 and test group 3 was significantly reduced, and the itching symptoms were effectively improved; further, compared with test group 1, the average number of scratching of mice in test group 3 was reduced from 5.11 times to 2.27 times, and the number of scratching was significantly reduced (p < 0.001), indicating that in the topical composition of the present invention, which is prepared by uniformly mixing the traditional Chinese medicine composition and the biological preparation in a weight ratio of 6:4, the biological preparation has an enhancing effect on the effect of the traditional Chinese medicine composition in relieving or treating skin itching symptoms, and the biological preparation plays the role of an effect enhancer / effect improver in the topical composition of the present invention.

[0162] Table 3 Serum neuropeptide concentration test results

[0163] Group SP (pg / mL) GRP (pg / mL) Histamine (ng / mL) Blank control group 22.35±1.19 50.24±3.83 0.26±0.05 Model Group 150.31±12.53 145.52±13.56 2.04±0.25 Positive control group <![CDATA[40.35±3.16 *** ]]> <![CDATA[63.24±4.36 *** ]]> <![CDATA[0.43±0.11 *** ]]> Experimental group 1 <![CDATA[82.10±5.32 *** ]]> <![CDATA[94.68±3.80 *** ]]> <![CDATA[0.83±0.10 *** ]]> Experimental Group 2 146.26±3.40 143.57±8.24 2.00±0.09 Experimental Group 3 <![CDATA[38.61±1.79 *** ]]> <![CDATA[61.28±5.98 *** ]]> <![CDATA[0.41±0.09 *** ]]>

[0164] Note: Compared with the model group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001; no significant difference was performed on the blank group, and there was no significant difference between the experimental group 2 and the model group (p > 0.05).

[0165] As shown in Table 3, compared with the blank control group, the concentrations of substance P (SP), gastrin-releasing peptide (GRP), and histamine in the blood of the model group mice increased abnormally after being sprayed with test substance A, 2,4-dinitrochlorophenylpropiophenone solution (1%).

[0166] Correspondingly, it can be seen from Table 2 that compared with the blank control group, the number of scratching times of mice in the model group was significantly increased, and the abnormally increased concentrations of substance P (SP), gastrin-releasing peptide (GRP), and histamine in the blood of mice were consistent with the increased trend of the number of skin itching attacks (scratching times) of mice.

[0167] As shown in Table 3, compared with the model group, in the experimental group 2 using the biological preparation prepared in Example 3, there was no significant change in the concentrations of SP, GRP and histamine in the serum of the experimental group 2.

[0168] Compared with the model group, the test group 1 using the topical composition prepared in Example 2 had blood SP, GRP, and histamine concentrations reduced by 45.38%, 34.94%, and 59.31%, respectively. This indicates that the application of the topical composition prepared in Example 2 of the present invention can reduce the concentrations of SP, GRP, and histamine in the blood. Accordingly, from Table 3 in combination with Table 2 and the comparison of the sensory state of the mouse skin, it can be seen that after the mice in the test group 1 were sprayed with the test substance B, which was the topical composition prepared in Example 2 of the present application, the changes in the sensory state of the mouse skin and the number of itching attacks (number of scratching) matched the trend of changes in the concentrations of SP, GRP, and histamine in the blood. This indicates that by applying the topical composition of the present invention to regulate the concentrations of neuropeptides (substance P, gastrin-releasing peptide) and histamine in the blood, the purpose of relieving or treating itching can be achieved, and it can be used as a skin repair agent.

[0169] Compared with the model group, the test group 3 used the external use composition prepared in Example 4, and the concentrations of SP, GRP, and histamine in the blood were reduced by 74.31%, 57.89%, and 79.90%, respectively; further, compared with the test group 1, the test group 3 used the external use composition prepared in Example 4, and the concentrations of SP, GRP, and histamine in the blood were reduced by 52.97%, 35.28%, and 50.60%, respectively, that is, relative to the test group 1, the reduction in SP, GRP, and histamine concentrations in the test group 3 was better; accordingly, from Table 3 in combination with Table 2 and the comparison of the sensory state of the mouse skin, it can be seen that after the mice in the test group 3 were sprayed with the test substance B, which was the external use composition prepared in Example 4 of the present application, their small The changes in the sensory state of mouse skin and the number of itching attacks (number of scratching) match the trend of changes in the concentrations of SP, GRP, and histamine in the blood, indicating that by applying the topical composition of the present invention to regulate the reduction of neuropeptide (substance P, gastrin-releasing peptide) and histamine concentrations in the blood, the purpose of relieving or treating itching can be achieved, and it can be used as a skin repair agent. At the same time, further, in the topical composition of the present invention prepared by uniformly mixing the traditional Chinese medicine composition and the biological preparation in a weight ratio of 6:4, the biological preparation has an enhancing effect on the effect of the traditional Chinese medicine composition in relieving or treating skin itching symptoms, and the biological preparation plays the role of an effect enhancer / effect improver in the topical composition of the present invention.

Claims

1. A composition for external use, characterized in that The external-use composition contains a traditional Chinese medicine composition, which includes Cnidium monnieri, Sophora flavescens, and Dictamnus chinensis root; The preparation method of the Chinese medicine composition comprises the following steps: Step (1): grinding each component of the traditional Chinese medicine composition separately and mixing them to prepare a mixture; Step (2): adding ethanol to the mixture obtained in step (1), soaking, filtering and collecting the filtrate to obtain the traditional Chinese medicine composition; The external composition is used in preparing a regulator for regulating the reduction of neuropeptide and / or histamine concentration in blood.

2. The external use composition according to claim 1, characterized in that The neuropeptides include substance P and / or gastrin-releasing peptide.

3. The external use composition according to claim 1, characterized in that The weight proportions of the components in the traditional Chinese medicine composition are as follows: 30 parts of Cnidium monnieri, 20 parts of Sophora flavescens, and 15 parts of Dictamnus cortex.

4. The external use composition according to claim 1, characterized in that The external composition further contains a biological agent; the biological agent comprises a fermentation product of Bacillus velezensis and / or a metabolite thereof.

5. The external use composition according to claim 4, characterized in that The Bacillus velezensis is Bacillus velezensis EYWS-B01, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 2025481.

6. The external use composition according to claim 4, characterized in that In the external-use composition, the weight ratio of the traditional Chinese medicine composition to the biological preparation is 6:4, and the external-use composition is obtained by uniformly mixing the two.

7. The external-use composition according to any one of claims 1 to 6, characterized in that The external-use composition is used in preparing medicines for relieving or treating itching, urticaria, eczema, and allergic dermatitis.

8. The external use composition according to claim 7, characterized in that The medicine is any one of a spray, a gel, a lotion, a cream, an emulsion, a paste, and an ointment.

9. The external use composition according to claim 8, characterized in that The medicine is a spray.

10. The external use composition according to claim 9, characterized in that Method of application of the drug: put the drug into a spray bottle.

Images