Compound essential oil for improving atopic dermatitis as well as preparation method and application of compound essential oil

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to compound essential oil for improving atopic dermatitis and a preparation method and application thereof. The compound essential oil comprises the following components in parts by mass: 1-5 parts of peppermint essential oil and 1-5 parts of eupatorium essential oil. The composition provided by the invention is applied to in-vivo efficacy verification in an atopic dermatitis mouse model, and the result shows that the compound essential oil has the effect of improving atopic dermatitis. The compound essential oil disclosed by the invention is safe and easily available in components, simple in preparation process, low in cost and good in curative effect, and can be used as a safe and effective external preparation for treating atopic dermatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a compound essential oil for improving atopic dermatitis, and a preparation method and application thereof. Background Art

[0002] Atopic dermatitis (AD) is a common inflammatory skin disease. It is a chronic, relapsing, and pruritic skin condition caused by an imbalanced immune response, genetic factors, and skin barrier dysfunction. It is characterized by recurring eczematous lesions and intense itching, often accompanied by symptoms such as redness, dryness, flaking, and erosion. Clinically, patients with atopic dermatitis often present with elevated serum IgE levels; abnormal expression of inflammatory cytokines and inflammatory cell infiltration in the skin; and skin barrier disruption manifested by increased transepidermal water loss, decreased bacterial diversity in the skin microbiome, and increased Staphylococcus aureus colonization. The disease affects people of all ages and races, but typically develops in early childhood between the ages of 3 and 6 months. Studies estimate that AD affects 15% to 30% of children and 2% to 10% of adults worldwide.

[0003] Currently, clinical treatment for AD primarily focuses on epidermal barrier repair, immune regulation, anti-inflammatory, antibacterial, and anti-allergic therapies. Western medicine often uses topical glucocorticoids, antihistamines, antibiotics, and immunomodulators. While these medications can quickly alleviate symptoms, long-term efficacy is poor, side effects are significant, and tolerance is difficult. Biologics, while effective, are expensive and beyond the reach of the average family. Therefore, developing AD medications with stable therapeutic effects, affordability, and minimal adverse reactions is of practical significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a compound essential oil for improving atopic dermatitis, a preparation method and an application thereof. The compound essential oil has the effect of improving atopic dermatitis.

[0005] The invention provides a compound essential oil for improving atopic dermatitis. The compound essential oil comprises the following components in parts by mass: 1 to 5 parts of peppermint essential oil and 1 to 5 parts of perilla essential oil.

[0006] As a preferred embodiment, the compound essential oil comprises the following components in parts by mass: 3 to 5 parts of peppermint essential oil and 1 part of perilla essential oil.

[0007] The present invention also provides a preparation method of the compound essential oil, comprising the following steps: respectively extracting peppermint essential oil and perilla essential oil by a steam distillation method, and mixing the peppermint essential oil and perilla essential oil to obtain the compound essential oil.

[0008] As a preferred solution, when preparing the peppermint essential oil, the mass volume ratio of the peppermint to water is 250-350 g:1600-2000 mL, and the steam distillation time is 4.5-5.5 h.

[0009] As a preferred solution, when preparing the perilla essential oil, the mass volume ratio of the perilla to water is 250-350 g:1600-2000 mL, and the steam distillation time is 4.5-5.5 h.

[0010] The present invention also provides application of the compound essential oil in preparing a medicine for treating atopic dermatitis.

[0011] As a preferred solution, the weight percentage of the compound essential oil in the preparation of the drug for treating atopic dermatitis is 0.5% to 2%.

[0012] As a preferred embodiment, the drug for treating atopic dermatitis is prepared from compound essential oil and excipients or carriers permitted for external use.

[0013] As a preferred embodiment, the preparation forms of the drug for treating atopic dermatitis include creams, ointments, liniments, gels and films.

[0014] As a preferred solution, the method of using the drug for treating atopic dermatitis includes application.

[0015] Beneficial Effects: The present invention provides a compound essential oil for improving atopic dermatitis. The compound essential oil comprises the following components by weight: 1-5 parts of peppermint essential oil and 1-5 parts of perilla essential oil. In vivo efficacy testing of the compound essential oil in an atopic dermatitis mouse model demonstrated that the compound essential oil improves atopic dermatitis. Furthermore, the compound essential oil is safe and readily available, has a simple preparation process, is low-cost, and exhibits excellent efficacy, making it a safe and effective topical preparation for the treatment of atopic skin inflammation.

[0016] This invention proposes for the first time that a mint and peony root compound essential oil has the effect of improving atopic dermatitis. Animal experiments confirm that the compound essential oil prepared using the present invention's formula can significantly alleviate skin lesions in mice with DNCB-induced atopic dermatitis, reduce dermatitis index scores, spleen index, and the frequency of scratching; reduce the thickness of the epidermis and dermis and mast cell infiltration in skin tissue; regulate the expression of inflammatory factors in mouse skin tissue; and improve skin barrier function. Furthermore, the present compound essential oil is derived from the common medicinal herbs mint and peony root through steam distillation. Its ingredients are safe and readily available, the preparation process is simple, the cost is low, and the efficacy is excellent, suggesting promising market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1 The antibacterial effects of different single essential oils on Staphylococcus aureus in Experimental Example 1 are shown, where a is mint; b is Ligusticum chuanxiong; c is Atractylodes macrocephala; d is Cyperus rotundus; e is Perilla frutescens; f is Patchouli chinensis; g is Cnidium monnieri; h is Artemisia capillaris; i is Acorus calamus; j is Chrysanthemum; k is Zanthoxylum bungeanum; l is Schizonepeta tenuifolia; m is Atractylodes macrocephala; n is Artemisia argyi; o is 5 mg / mL neomycin sulfate;

[0019] Figure 2 The following are the statistics of the diameters of the inhibition zones of different single essential oils against Staphylococcus aureus in Experimental Example 1, where a is mint; b is Ligusticum chuanxiong; c is Atractylodes macrocephala; d is Cyperus rotundus; e is Perilla frutescens; f is Patchouli chinensis; g is Cnidium monnieri; h is Artemisia capillaris; i is Acorus calamus; j is Chrysanthemum; k is Zanthoxylum bungeanum; l is Schizonepeta tenuifolia; m is Atractylodes macrocephala; n is Artemisia argyi; o is 5 mg / mL neomycin sulfate;

[0020] Figure 3 The antibacterial effects of different compound essential oils on Staphylococcus aureus in Experimental Example 1 are shown, where a is F1 (mint: perilla: ligusticum: artemisia argyi); b is F2 (mint: perilla: ligusticum); c is F3 (mint: perilla: artemisia argyi); d is F4 (mint: ligusticum: artemisia argyi); e is F5 (perilla: ligusticum: artemisia argyi); f is F6 (mint: perilla); g is F7 (mint: ligusticum); h is F8 (mint: artemisia argyi); i is F9 (perilla: ligusticum); j is F10 (perilla: artemisia argyi); k is F11 (ligusticum: artemisia argyi); l is 5 mg / mL neomycin sulfate, and the proportions of the essential oils are all equal in mass ratio;

[0021] Figure 4 The statistics of the inhibition zone diameters of different compound essential oils against Staphylococcus aureus in Experimental Example 1, where a is F1 (mint: perilla: ligusticum: artemisia argyi); b is F2 (mint: perilla: ligusticum); c is F3 (mint: perilla: artemisia argyi); d is F4 (mint: ligusticum: artemisia argyi); e is F5 (perilla: ligusticum: artemisia argyi); f is F6 (mint: perilla); g is F7 (mint: ligusticum); h is F8 (mint: artemisia argyi); i is F9 (perilla: ligusticum); j is F10 (perilla: artemisia argyi); k is F11 (ligusticum: artemisia argyi); l is 5 mg / mL neomycin sulfate, and the proportions of each essential oil are all equal in mass ratio;

[0022] Figure 5The cytotoxicity and NO inhibition rate of different single essential oils on RAW264.7 cells in Experimental Example 2, where A is the cell survival rate and B is the NO inhibition rate of different plant essential oils; a is mint; b is Ligusticum chuanxiong; c is Atractylodes lancea; d is Cyperus rotundus; e is Perilla frutescens; f is Patchouli; g is Cnidium monnieri; h is Artemisia capillaris; i is Acorus calamus; j is Chrysanthemum; k is Zanthoxylum bungeanum; l is Schizonepeta tenuifolia; m is Atractylodes macrocephala; n is Artemisia argyi; o is dexamethasone;

[0023] Figure 6 EC of peppermint essential oil, perilla essential oil, atractylodes essential oil, ligusticum essential oil, perilla essential oil and patchouli essential oil in Experimental Example 2 50 value;

[0024] Figure 7 The cytotoxicity and NO inhibition rate of different compound essential oils on RAW264.7 cells in Experimental Example 2, where A is the cell survival rate and B is the NO inhibition rate of different compound essential oils;

[0025] Figure 8 is the EC of different compound essential oils in Experimental Example 2 50 value;

[0026] Figure 9 The cytotoxicity of essential oils in different ratios to RAW264.7 cells in Experimental Example 3;

[0027] Figure 10 The effect of the compound essential oil cream in Experimental Example 4 on the body weight, skin morphology and dermatitis score of AD mice, where A is the body weight statistics, B is the dermatitis severity score, and C is the skin morphology;

[0028] Figure 11 The effect of the compound essential oil cream in Experimental Example 4 on the spleen index and scratching frequency of AD mice, where A is the spleen index and B is the scratching frequency;

[0029] Figure 12 The results of HE staining of the compound essential oil cream on the back skin lesions of AD mice in Experimental Example 4 are shown. The left figure is HE staining, and the right figure (A) is the epidermal thickness and (B) is the dermal thickness. The black bar represents the scale, and the length represents 200 μm.

[0030] Figure 13 The results of the effect of the compound essential oil cream on TB staining of the back lesions of AD mice in Experimental Example 4 are shown. The left figure shows the TB staining results, and the right figure shows the number of mast cells. The black bar represents the scale, and the length represents 200 μm.

[0031] Figure 14The expression of inflammatory cytokine mRNA in the back skin tissue of AD mice treated with the compound essential oil cream in Experimental Example 4 is shown in Figure 4, where A is the relative expression of iNOS mRNA, B is the relative expression of TNF-α mRNA, C is the relative expression of IL-1β mRNA, D is the relative expression of IL-4 mRNA, E is the relative expression of IL-6 mRNA, and F is the relative expression of IL-31 mRNA;

[0032] Figure 15 The expression of skin barrier-related proteins mRNA and water loss in the back skin of AD mice by the compound essential oil cream in Experimental Example 4 are shown in Figure 4, where A is the TEWL of the back skin of mice, B is the relative expression of Occludin mRNA, C is the relative expression of Claudin-1 mRNA, and D is the relative expression of Loricrin mRNA. DETAILED DESCRIPTION

[0033] The present invention provides a compound essential oil for improving atopic dermatitis, comprising the following components by mass: 1 to 5 parts of peppermint essential oil and 1 to 5 parts of cyperus rotundus essential oil. As a specific embodiment, the efficacy of each Chinese herbal essential oil was evaluated by comprehensive scoring of antibacterial and anti-inflammatory activity indicators. It was found that the diameter of the antibacterial zone of peppermint and cyperus rotundus was greater than 15 mm, and they were highly sensitive to Staphylococcus aureus. The antibacterial effect of both was better than that of 5 mg / mL penicillin sulfate. The inhibition rate of NO in RAW264.7 cells induced by LPS was EC 0. 50 They were 28.72 and 42.00 μg / mL respectively, showing excellent anti-inflammatory effects.

[0034] As a specific embodiment, the mass proportions of the compound essential oil can be 1 part of peppermint essential oil and 1 part of perilla essential oil, 2 parts of peppermint essential oil and 1 part of perilla essential oil, 3 parts of peppermint essential oil and 1 part of perilla essential oil, 4 parts of peppermint essential oil and 1 part of perilla essential oil, 5 parts of peppermint essential oil and 1 part of perilla essential oil, 1 part of peppermint essential oil and 5 parts of perilla essential oil, 1 part of peppermint essential oil and 4 parts of perilla essential oil, 1 part of peppermint essential oil and 3 parts of perilla essential oil or 1 part of peppermint essential oil and 2 parts of perilla essential oil; as a specific embodiment, when the mass proportions of the compound essential oil are 3 parts of peppermint essential oil and 1 part of perilla essential oil, 4 parts of peppermint essential oil and 1 part of perilla essential oil or 5 parts of peppermint essential oil and 1 part of perilla essential oil, it has more outstanding antibacterial and anti-inflammatory activity, proving that when the mass proportions of the compound essential oil are 3 to 5 parts of peppermint essential oil and 1 part of perilla essential oil, it is the best ratio of the compound essential oil.

[0035] As a specific embodiment, the compound essential oil of the present invention is a main component of aromatic plants, has a pungent taste, and can open the pores. When administered through the skin, it can achieve the effect of external treatment of internal diseases. In addition, the compound essential oil of the present invention is a small molecule substance, is hydrophobic, and has good transdermal penetration promoting effect. It can be absorbed through the skin and mucous membranes and enter the blood circulation and systemic circulation. External application of the essential oil can promote wound healing, repair skin and mucous membranes, and has a good inhibitory effect on skin inflammation and allergic reactions.

[0036] The present invention also provides a preparation method of the compound essential oil, comprising the following steps: respectively extracting peppermint essential oil and perilla essential oil by a steam distillation method, and mixing the peppermint essential oil and perilla essential oil to obtain the compound essential oil.

[0037] When preparing the peppermint essential oil of the present invention, the mass volume ratio of the peppermint to water is 250-350 g:1600-2000 mL, and the steam distillation time is 4.5-5.5 h; as a specific embodiment, the mass volume ratio of the peppermint to water can be 250 g:1600 mL, 250 g:1700 mL, 250 g:1800 mL, 250 g:1900 mL, 250 g:2000 mL, 260 g:1600 mL, 260 g:1700 mL, 260 g:1800 mL, 260 g:1900 mL, 260 g:2000 mL, 280 g:1600 mL, 280 g:1700 mL, 280 g:1800 mL, 280 g:1900 mL, 280 g: As a specific embodiment, the steam distillation time can be 4.5h, 4.6h, 4.7h, 4.8h, 4.9h, 5.0h, 5.1h, 5.2h, 5.3h, 5.4h or 5.5h.

[0038] When preparing the perilla essential oil of the present invention, the mass volume ratio of the perilla to water is 250-350g:1600-2000mL, and the steam distillation time is 4.5-5.5h; as a specific embodiment, the mass volume ratio of the perilla to water can be 250g:1600mL, 250g:1700mL, 250g:1800mL, 250g:1900mL, 250g:2000mL, 260g:1600mL, 260g:1700mL, 260g:1800mL, 260g:1900mL, 260g:2000mL, 280g:1600mL, 280g:1700mL, 280g:1800mL, 280g:1900mL, 280g: As a specific embodiment, the steam distillation time can be 4.5h, 4.6h, 4.7h, 4.8h, 4.9h, 5.0h, 5.1h, 5.2h, 5.3h, 5.4h or 5.5h.

[0039] The present invention also provides use of the compound essential oil in preparing a drug for treating atopic dermatitis; the weight percentage of the compound essential oil in the drug for treating atopic dermatitis is 0.5% to 2%; as a specific embodiment, the weight percentage of the compound essential oil in the drug for treating atopic dermatitis can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%.

[0040] In one embodiment, the atopic dermatitis treatment drug is prepared from a compound essential oil and an excipient or carrier acceptable for topical use. In one embodiment, the atopic dermatitis treatment drug is available in formulations including creams, ointments, liniments, gels, and films. In one embodiment, the atopic dermatitis treatment drug is administered by topical application.

[0041] As a specific embodiment, the compound essential oil cream prepared by the present invention can significantly alleviate the skin lesions of DNCB-induced atopic dermatitis, reduce the dermatitis index score, reduce the spleen index and reduce the number of scratching; reduce the thickness of the epidermis and dermis of skin tissue and the infiltration of mast cells; regulate the expression of inflammatory factors in mouse skin tissue; and improve skin barrier function.

[0042] To further illustrate the present invention, the following detailed description of a compound essential oil for improving atopic dermatitis, its preparation method, and application is provided by the present invention in conjunction with examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0043] Unless otherwise specified, the present invention has no special requirements for the raw materials, and commercially available products known to those skilled in the art can be used.

[0044] Example 1 Preparation of essential oil

[0045] Refer to the 2020 edition of the "Chinese Pharmacopoeia" Part IV 2204 General Rules for the Determination of Volatile Oils Method A to extract the essential oils of mint, Ligusticum chuanxiong, Atractylodes lancea, Cyperus rotundus, Perilla frutescens, Patchouli, Cnidium monnieri, Artemisia capillaris, Acorus calamus, chrysanthemum, Zanthoxylum bungeanum, Schizonepeta tenuifolia, Atractylodes macrocephala and Artemisia argyi.

[0046] Extraction Method: Grind the test herbs and pass them through a No. 2 sieve. Weigh 300g of each herbal powder into a 5000mL round-bottom flask. Add 1800mL of distilled water and shake to mix. Connect a volatile oil analyzer to a reflux condenser. Add water from the top of the condenser until the scale on the volatile oil analyzer is filled and overflows into the flask. Heat in a heating mantle until boiling and maintain a slight boil for approximately 5 hours, until the amount of oil in the analyzer no longer increases. Stop heating and collect the essential oils of each Chinese herbal medicine.

[0047] Embodiment 2 Peppermint Perilla compound essential oil

[0048] Essential oil was prepared according to the method of Example 1, and 4 parts of peppermint essential oil and 1 part of perilla essential oil were mixed to prepare a compound essential oil. The compound essential oil and pharmaceutical carrier were prepared into an oil-in-water cream according to the weight percentage of the components in Table 1. The pharmaceutical carrier components, by weight, were as follows: 0.8 parts of octadecanol (Batch No. 20210325), 2.5 parts of stearic acid (Batch No. 20220808), 3.5 parts of liquid paraffin (Batch No. 20230419), 0.5 parts of white vaseline (Batch No. 20220525), 3 parts of glycerin (Batch No. 20240329), 0.8 parts of triethanolamine (Batch No. 20240315), and 0.1 parts of ethyl paraben (Batch No. 20241010) purchased from Sinopharm Chemical Reagent Co., Ltd. 1.6, 1.4, or 1 parts of olive oil (Article No. O815211) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. and 27 parts of water. Store in a sealed container away from light for future use.

[0049] Table 1 Component content in each embodiment

[0050]

[0051] Experimental Example 1 In vitro antibacterial experiment

[0052] 1. Experimental strains and test drugs

[0053] The experimental strain was Staphylococcus aureus (ATCC6538), which was purchased from Beijing Baozang Biotechnology Co., Ltd.

[0054] The test drugs used in the experiment were the single essential oils in Example 1 (mint, Ligusticum chuanxiong, Atractylodes lancea, Cymbidium orientale, Perilla leaf, Patchouli, Cnidium monnieri, Artemisia capillaris, Acorus calamus, chrysanthemum, Zanthoxylum bungeanum, Schizonepeta tenuifolia, Atractylodes macrocephala and Artemisia argyi) or compound essential oils prepared in equal proportions of the single essential oils: F1 (mint: Cymbidium orientale: Ligusticum chuanxiong: Artemisia argyi); F2 (mint: Cymbidium orientale: Ligusticum chuanxiong); F3 (mint: Cymbidium orientale: Artemisia argyi); F4 (mint: Ligusticum chuanxiong: Artemisia argyi); F5 (Cymbidium orientale: Ligusticum chuanxiong: Artemisia argyi); F6 (mint: Cymbidium orientale); F7 (mint: Ligusticum chuanxiong); F8 (mint: Artemisia argyi); F9 (Cymbidium orientale: Ligusticum chuanxiong); F10 (Cymbidium orientale: Artemisia argyi); F11 (Ligusticum chuanxiong: Artemisia argyi), and the proportions of the essential oils were all equal in mass ratio.

[0055] The positive control drug was neomycin sulfate, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 5.0 mg of neomycin sulfate was accurately weighed using an analytical balance and dissolved in 1 mL of sterile distilled water to prepare a 5 mg / mL neomycin sulfate solution.

[0056] 2. Experimental Methods

[0057] (1) Preparation of culture medium

[0058] Dissolve 3.0 g beef extract, 10.0 g tryptone, 5.0 g sodium chloride, and 20.0 g agar in distilled water to 1.0 L. Adjust the pH to 7.4 with 1 mol / L NaOH and sterilize by autoclaving at 121°C for 20 min. The liquid medium does not contain agar, but remains the same.

[0059] (2) Preparation of bacterial solution

[0060] Inoculate Staphylococcus aureus onto solid culture medium and incubate at 37°C for 24 hours. Use an inoculation loop to pick a single colony and inoculate it into 2.5 mL of liquid culture medium. Incubate at 37°C with shaking for 24 hours. Use the plate streak method to inoculate the bacteria onto the surface of the solid culture medium and continue incubating at 37°C for another 24 hours. Pick a single colony of Staphylococcus aureus and dilute the bacterial solution to 1×10 8 CFU / mL standard concentration was used to prepare the test bacterial solution.

[0061] (3) Inhibition zone test

[0062] The inhibition zone test was performed using the filter paper method. A sterile cotton swab was dipped into the test bacterial solution and evenly smeared onto a solid plate. After a short period of smearing, a 6mm sterile blank drug-sensitive sheet was placed on the plate containing the bacteria. Using a pipette, 5μL of each test drug was dripped onto the blank sheet. The sheet was then incubated upside down at 37°C for 24 hours. Colony growth was observed and the diameter of the inhibition zone was measured using the cross-hatch method. Three replicates were performed for each drug.

[0063] 3. Experimental Results

[0064] (1) Antibacterial effects of different essential oils on Staphylococcus aureus

[0065] Figure 1 and Figure 2 The results showed that the inhibitory effects of the 14 single essential oils on Staphylococcus aureus varied significantly. Peppermint, Perilla frutescens, and Artemisia argyi essential oils showed the greatest antibacterial activity, with inhibition zone diameters exceeding 15 mm, indicating high sensitivity to S. aureus. Furthermore, peppermint and Perilla frutescens essential oils exhibited greater antibacterial activity than 5 mg / mL penicillin sulfate, while Artemisia argyi was comparable to penicillin sulfate.

[0066] (2) Antibacterial effects of different compound essential oils on Staphylococcus aureus

[0067] Based on the in vitro antibacterial and anti-inflammatory activities of single plant essential oils, 11 essential oils from the top three medicinal plants (menthol, perilla, ligusticum, and artemisia) were selected and formulated in equal proportions. The efficacy was evaluated based on the combined scores of antibacterial and anti-inflammatory activities.

[0068] Depend on Figure 3 and Figure 4 It can be seen that F6 (mint + perilla) showed the best antibacterial activity, with an inhibition zone size of 28.48 mm for inhibiting Staphylococcus aureus, which was better than 26.38 mm for single mint essential oil and 24.06 mm for perilla essential oil, and the antibacterial effect was enhanced.

[0069] Experimental Example 2 In vitro anti-inflammatory experiment

[0070] 1. Experimental Materials and Reagents

[0071] Mouse RAW264.7 macrophages (Cat. No. CL-0190) were purchased from Wuhan Punosai Life Science Technology Co., Ltd.; MTT kit (Cat. No. ST1537) and NO kit (Cat. No. ST1537) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; lipopolysaccharide LPS (Cat. No. L8880) was purchased from Solebo Biotechnology Co., Ltd.; dexamethasone DXMS (Cat. No. S17003) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0072] The test drugs used in the experiment were the single essential oils in Example 1 (mint, Ligusticum chuanxiong, Atractylodes lancea, Cymbidium orientale, Perilla leaf, Patchouli, Cnidium monnieri, Artemisia capillaris, Acorus calamus, chrysanthemum, Zanthoxylum bungeanum, Schizonepeta tenuifolia, Atractylodes macrocephala and Artemisia argyi) or compound essential oils prepared in equal proportions of the single essential oils: F1 (mint: Cymbidium orientale: Ligusticum chuanxiong: Artemisia argyi); F2 (mint: Cymbidium orientale: Ligusticum chuanxiong); F3 (mint: Cymbidium orientale: Artemisia argyi); F4 (mint: Ligusticum chuanxiong: Artemisia argyi); F5 (Cymbidium orientale: Ligusticum chuanxiong: Artemisia argyi); F6 (mint: Cymbidium orientale); F7 (mint: Ligusticum chuanxiong); F8 (mint: Artemisia argyi); F9 (Cymbidium orientale: Ligusticum chuanxiong); F10 (Cymbidium orientale: Artemisia argyi); F11 (Ligusticum chuanxiong: Artemisia argyi), and the proportions of the essential oils were all equal in mass ratio.

[0073] 2. Experimental Methods

[0074] (1) Cell culture

[0075] RAW264.7 cells were cultured at 37°C in a humidified environment of 5% CO2 using DMEM medium containing 10% fetal bovine serum and 1.0% penicillin-streptomycin solution.

[0076] (2) Cell viability assay

[0077] The cytotoxicity of different essential oils on LPS-stimulated RAW264.7 cells was detected by MTT assay (refer to the instructions of MTT kit). 5 Cells were seeded at a density of 1 μg / mL in a 96-well plate and incubated for 24 hours. LPS (final concentration 1 μg / mL) and essential oils at varying final concentrations (5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL) were then added to each well. A blank control group containing only solvent and cells was also incubated for 24 hours. 10 μL of 5 mg / mL L-MTT solution was added to each well and incubated for 4 hours. The solution in the 96-well plate was discarded, and 200 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance at 490 nm was measured in triplicate for each group.

[0078] Cell survival rate = [OD(drug group)-OD(blank group)] / [OD(control group)-OD(blank group)]×100%

[0079] (3) NO determination

[0080] Adjust the cell density to 6 × 10 5 / mL, inoculated into 96-well plates and cultured for 24h. Set up blank group, control group (1μg / mL LPS), positive drug DXMS group (5, 10, 20, 40 and 80μg / mL DXMS + 1μg / mL LPS), essential oil treatment group (5, 10, 20, 40 and 80μg / mL essential oil + 1μg / mL LPS), each well of 200μL, stimulate cells for 24h, then aspirate 100μL supernatant, add Griess Reagent according to the NO kit, shake for 10min, and measure absorbance at 540nm. Each group is set up in triplicate.

[0081] NO inhibition rate = 1-{[OD(drug group)-OD(blank group)] / [OD(control group)-OD(blank group)]}×100%

[0082] 3. Experimental Results

[0083] (1) Effects of different essential oils on NO content in LPS-induced RAW264.7 cells

[0084] Figure 5 Figure A shows that except for the essential oils of Artemisia capillaris and Atractylodes macrocephala, which showed cytotoxicity at 80 μg / mL, the other 12 essential oils and dexamethasone had no obvious toxic side effects on RAW264.7 cells and were safe to use within this concentration range. Figure 5 Figure B shows that different plant essential oils exhibit different degrees of inhibitory effect on NO release at concentrations of 5 to 80 μg / mL. Among them, the essential oils with NO inhibition rates exceeding 50% are peppermint, perilla, atractylodes, ligusticum, perilla leaves, and patchouli. Figure 6 It can be seen that the EC of different plant essential oils on NO inhibition rate 50 As the basis for evaluation, the results showed that mint, ligusticum chuanxiong and perilla had the best anti-inflammatory effects, EC 50 They were 28.72, 38.20 and 42.00 μg / mL respectively.

[0085] (2) Effects of different essential oil compounds on NO content in LPS-induced RAW264.7 cells

[0086] Figure 7 Figure A shows that the survival rates of RAW264.7 cells were all greater than 90% at concentrations of 5 to 80 μg / mL for the 11 compound essential oils, indicating that the drugs were safe and non-toxic within this dosage range. Figure 7 Middle B and Figure 8 It can be seen that the NO inhibition rate of each compound essential oil except F10 is greater than 50% at 80 μg / mL. Among them, F2, F6, F7 and F8 showed better anti-inflammatory activity than single essential oils, and the EC 50were 23.36, 24.99, 21.22 and 28.49 μg / mL, respectively, and the anti-inflammatory effect was enhanced.

[0087] Combined with the results of Experimental Example 1, from Table 2 (the score is based on the inhibition zone and NO inhibition rate EC 50 The compound with the largest inhibition zone diameter among the 11 compounds was scored 11 points, and the compound with the smallest inhibition zone diameter was scored 1 point; EC 50 The minimum is 11 points, EC 50 The largest is 1 point) It can be seen that F6 (mint + perilla) has the highest comprehensive score, which is better than the effect of any single prescription, and plays a synergistic role in antibacterial and anti-inflammatory activities. Therefore, mint and perilla compound essential oil was selected for subsequent research.

[0088] Table 2 Results of efficacy scores of 11 compound essential oils

[0089]

[0090]

[0091] Experimental Example 3 Evaluation of the antibacterial and anti-inflammatory activities of mint and perilla compound essential oils in different ratios

[0092] Referring to the methods of Experimental Examples 1 and 2, the inhibition zone diameters of the compound essential oils of mint and perilla essential oils in different ratios (1:1, 2:1, 3:1, 4:1, 5:1, 1:5, 1:4, 1:3, 1:2) against Staphylococcus aureus and the NO inhibition rate at a dose of 80 μg / mL were determined. Figure 9 The results showed that 80 μg / mL of compound essential oils in different ratios (B represents peppermint and P represents cyperus rotundus, for example, 4B:1P refers to a mixture of 4 parts peppermint and 1 part cyperus rotundus) were nontoxic to RAW264.7 cells. Table 3 shows that peppermint:cyperus rotundus ratios of 3:1, 4:1, and 5:1 exhibited superior antibacterial and anti-inflammatory activity. Therefore, the present formulation consists of 3-5 parts peppermint essential oil and 1 part cyperus rotundus essential oil.

[0093] Table 3 Antibacterial and anti-inflammatory activities of peppermint and perilla essential oils in different ratios

[0094] sample Diameter of inhibition zone (mm) NO inhibition rate (%) 1B:1P 26.03±1.17 93.79±4.06 2B:1P 26.22±2.04 94.72±1.51 3B:1P 27.19±0.91 95.03±4.22 4B:1P 29.85±0.72 95.50±1.18 5B:1P 28.46±2.16 95.03±1.98 1B: 5P 28.39±1.16 94.10±4.30 1B:4P 27.84±1.01 90.99±2.01 1B:3P 26.72±1.30 91.77±4.37 1B:2P 26.66±1.65 91.27±4.99

[0095] Experimental Example 4 Animal Experiment

[0096] In this experimental example, the compound essential oil cream preparation prepared in Example 2 was used as a topical preparation. By establishing an atopic dermatitis animal model in mice, the therapeutic effects of the compound essential oil preparation prepared by the present invention and 0.05% dexamethasone cream were compared, in order to develop a natural and effective topical preparation for improving atopic dermatitis.

[0097] 1. Experimental Grouping

[0098] Forty-eight SPF female Balb / c mice aged 6 to 8 weeks were randomly divided into 6 groups, namely, blank control group, atopic dermatitis model group, dexamethasone cream positive drug group, 0.5% compound essential oil cream low-dose group, 1% compound essential oil cream medium-dose group and 2% compound essential oil cream high-dose group, with 8 mice in each group.

[0099] 2. Experimental Methods

[0100] After one week of acclimatization, the mice were depilated on their backs using an electric shaver and depilatory cream on the day before the experiment, covering an area of 2 cm × 2 cm. 2,4-Dinitrochlorobenzene (DNCB) (Sigma-Aldrich Trading Co., Ltd., Catalog No. 138630) was diluted to 1% and 0.4% in acetone (Nanjing Chemical Reagent Co., Ltd., Lot No. 240823175F) and olive oil (3:1, v / v). In the first week, the dorsal skin of each treatment group and the model group was sensitized with 100 μL of 1% DNCB twice a week. In the second, third, and fourth weeks, 100 μL of 0.4% DNCB was applied to the dorsal skin to maintain AD symptoms three times a week. The blank control group was treated with the same amount of solvent. Starting on day 7 of the experiment, 0.5%, 1%, or 2% compound essential oil cream or dexamethasone cream was applied daily to the dorsal skin of the corresponding mice. The blank control and model groups were treated with blank cream base for three weeks. After the 4-week experimental period, spleen and skin tissues were collected on the next day (day 29).

[0101] 3. Experimental indicators

[0102] The weight changes of mice were recorded on days 0, 7, 14, 21, and 28 after the start of the experiment, and the severity of DNCB-induced AD lesions was scored according to Table 4. On the day of the end of the experiment, dermatoscopy was performed, the number of scratches of the mice within 10 minutes was counted, and the transepidermal water loss (TEWL) of the shaved area on the back was measured using a skin physiological detector MPA580. The spleen index was calculated [spleen index = spleen weight (g) / body weight (g) × 100%]; and the hematoxylin-eosin staining method (H&E staining) was used to observe the lesions. The changes in the thickness of the epidermis and dermis and the number of inflammatory cells in the skin tissue were observed; the changes in the number of mast cells in the skin tissue were observed by toluidine blue staining (TB staining) (Wang Yanjie. Study on the effect and mechanism of bisdemethoxycurcumin on atopic dermatitis [D]. Jilin University, 2022.DOI:10.27162 / d.cnki.gjlin.2022.003534.); the mRNA expression of inflammatory factors iNOS, TNF-α, IL-1β, IL-4, IL-6 and IL-31 and skin barrier-related proteins Occludin, Claudin-1 and Loricrin in the skin tissue were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The primer sequences are shown in Table 5.

[0103] Table 4 Skin dermatitis degree scoring table

[0104] Score erythema edema scales Exfoliation 0 No erythema No edema No scales No skin peeling 1 Small red spots are faintly visible Mild edema that does not rise above the skin surface bran-like scales Slight skin peeling 2 Clearly visible medium-sized erythema Moderate edema above the skin surface flaky scales Partial epidermal exfoliation 3 Obvious large erythema Severe edema that is significantly higher than the skin surface Large, thick scales Extensive epidermal exfoliation

[0105] Table 5 Primer sequences

[0106]

[0107]

[0108] 4. Statistical methods

[0109] All experimental data were expressed as mean ± standard deviation and statistically processed using GraphPad Prism 8. One-way ANOVA was used to compare multiple groups, and P < 0.05 was considered statistically significant.

[0110] 5. Experimental Results

[0111] (1) Effects of compound essential oil cream on body weight and dermatitis severity in AD mice

[0112] Depend on Figure 10As shown in Figure A, after the application of DNCB, the weight of mice in each group decreased compared with the blank group. After treatment, the weight of mice in the compound essential oil treatment groups with different doses was restored and approached that of the blank group, while the weight of mice in the positive group decreased significantly. Figure 10 Figure B shows that in the mouse dermatitis severity score, treatment with different doses of compound essential oil cream reduced the dermatitis index compared with the DNCB model group. Representative images of skin lesions in each group of mice after treatment are shown in Figure 2. Figure 10 As shown in Figure C, consistent with the dermatitis index score, repeated application of DNCB induced significant AD-like clinical symptoms in mice, including skin erythema, edema, scaling, erosions, and excoriations. However, topical treatment with the essential oil cream and dexamethasone cream reduced visible skin lesions. These observations clearly indicate that the essential oil cream alleviated AD-like symptoms in mice, with the most significant improvement observed in the 2% high-dose group.

[0113] (2) Effects of compound essential oil cream on spleen index and scratching frequency in AD mice

[0114] After continuous stimulation with DNCB, the spleen index of the AD model mice increased significantly, with significant differences compared to the blank group (p<0.001). After treatment, the spleen index of each drug group decreased to a certain extent, among which the spleen index of the mice in the high-dose compound essential oil cream group and the positive drug group decreased significantly ( Figure 11 A). Skin itching is a typical feature of atopic dermatitis, so the number of times each group of mice scratched their back skin within 10 minutes after the treatment cycle was completed was counted to investigate the antipruritic effect of the compound essential oil cream. Figure 11 As shown in Figure B, compared with the blank group, the scratching frequency of mice in the AD model group increased significantly, and the scratching behavior of mice in each group was alleviated after treatment. Among them, the scratching frequency of mice in the medium and high dose groups of the compound essential oil cream was significantly reduced compared with the model group.

[0115] (3) Effect of compound essential oil cream on HE staining of back lesions in AD mice

[0116] HE staining results Figure 12As shown in the blank group, the epidermis and dermis were intact, the dermal collagen was regular, and only a few inflammatory cells such as lymphocytes and neutrophils infiltrated. There was no edema, necrosis, or keratinization in the tissue. Compared with the blank group, the AD model group showed significant thickening of the epidermis and dermis, sloughed scabs, and extensive infiltration of inflammatory cells such as neutrophils and lymphocytes in the dermis. Compared with the AD model group, the epidermal thickness of mice in each treatment group was significantly reduced, and the differences were significant (p < 0.001). In addition, the dermal thickness of mice in the medium-dose and high-dose groups of the compound essential oil cream was significantly reduced, and mild dermal inflammatory cell infiltration was observed. This proves that the compound essential oil cream can improve the pathological thickening of the skin and the infiltration of inflammatory cells to a certain extent.

[0117] (4) Effect of compound essential oil cream on TB staining of back lesions in AD mice

[0118] Toluidine blue staining was used to evaluate the skin mast cell infiltration ( Figure 13 Compared with the blank group, the skin of the AD model mice showed an abnormal increase in the number of mast cells and significant infiltration (p<0.001). Mast cell infiltration was significantly improved in all treated groups, and the number of mast cells was significantly reduced.

[0119] (5) Effect of compound essential oil cream on mRNA expression of inflammatory cytokines in back lesions of AD mice

[0120] Various inflammatory cytokines are involved in the inflammatory response in the pathogenesis of AD. Figure 14 It can be seen that compared with the blank group, DNCB stimulation significantly increased the mRNA expression of iNOS, TNF-α, IL-1β, IL-4, IL-6, and IL-31 in the skin lesions of mice in the model group. Compared with the model group, the mRNA expression of iNOS, TNF-α, IL-1β, IL-4, IL-6, and IL-31 in the skin lesions of mice in each treatment group showed varying degrees of decrease. Among them, different doses of the compound essential oil cream significantly reduced the expression of IL-1β mRNA (p < 0.001). The medium-dose and high-dose groups of the compound essential oil cream significantly reduced the expression levels of iNOS, TNF-α, IL-4, IL-6, and IL-31, and all were statistically significant. This proves that the compound essential oil cream can inhibit the inflammatory response by reducing the mRNA expression of inflammatory cytokines.

[0121] (6) Effects of compound essential oil cream on skin barrier indices of back lesions in AD mice

[0122] The development of atopic dermatitis is often accompanied by abnormalities in the skin barrier. TEWL is an important indicator for judging skin barrier function. Therefore, the mRNA expression of TEWL in mouse skin tissue and skin barrier-related proteins in the lesion area was measured to explore the effect of the compound essential oil cream on the skin barrier. Figure 15 ). Compared with the blank group, the TEWL of mice in the AD model group was significantly increased (p<0.001). Compared with the AD model group, the TEWL of mice in the medium-dose and high-dose groups of the compound essential oil cream was significantly reduced (p<0.01 and p<0.001), while the improvement in the low-dose group and the positive drug group of the compound essential oil cream was poor and not statistically significant. In the expression detection of skin barrier protein mRNA, DNCB stimulation caused the expression levels of Occludin, Claudin-1 and Loricrin mRNA in the model group mice to be significantly reduced (p<0.001). Compared with the model group, the expression of skin barrier protein mRNA in each treatment group was increased, among which the high-dose group of the compound essential oil significantly increased the expression levels of Occludin, Claudin-1 and Loricrin mRNA, and had the best effect on improving the skin barrier.

[0123] It can be seen that the compound essential oil cream prepared by the present invention can significantly alleviate the skin lesions of DNCB-induced atopic dermatitis, reduce the dermatitis index score, reduce the spleen index and reduce the number of scratching; reduce the thickness of the epidermis and dermis of skin tissue and the infiltration of mast cells; regulate the expression of inflammatory factors in mouse skin tissue; and improve skin barrier function.

[0124] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A compound essential oil for improving atopic dermatitis, characterized in that: The compound essential oil comprises the following components in parts by mass: 1 to 5 parts of peppermint essential oil and 1 to 5 parts of perilla essential oil.

2. The compound essential oil according to claim 1, wherein The compound essential oil comprises the following components in parts by mass: 3 to 5 parts of peppermint essential oil and 1 part of perilla essential oil.

3. The method for preparing the compound essential oil according to claim 1 or 2, wherein The following steps are involved: Peppermint essential oil and peony root essential oil are extracted respectively by a steam distillation method, and the peppermint essential oil and peony root essential oil are mixed to obtain the compound essential oil.

4. The preparation method according to claim 3, characterized in that When preparing the peppermint essential oil, the mass volume ratio of the peppermint to water is 250-350 g:1600-2000 mL, and the steam distillation time is 4.5-5.5 h.

5. The preparation method according to claim 3, characterized in that When preparing the perilla essential oil, the mass volume ratio of the perilla to water is 250-350 g:1600-2000 mL, and the steam distillation time is 4.5-5.5 h.

6. Use of the compound essential oil according to claim 1 or 2 or the compound essential oil prepared by the preparation method according to any one of claims 3 to 5 in the preparation of a medicament for treating atopic dermatitis.

7. The use according to claim 6, characterized in that The weight percentage of the compound essential oil in the preparation of the medicine for treating atopic dermatitis is 0.5% to 2%.

8. The use according to claim 6, characterized in that The drug for treating atopic dermatitis is prepared from compound essential oil and excipients or carriers permitted by external medicines.

9. The use according to claim 6, characterized in that The preparation forms of the drug for treating atopic dermatitis include creams, ointments, liniments, gels and films.

10. The use according to claim 6, characterized in that The method of using the drug for treating atopic dermatitis includes application.

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