Application of linalool in preparation of medicine for treating atopic dermatitis

Abstract

The invention relates to the field of new application of compounds, and particularly provides application of linalool in preparation of a medicine for treating atopic dermatitis. Research finds that the natural chemical component linalool can relieve AD symptoms and regulate skin microbiota through the following mechanisms: key inflammatory mediators (such as IgE, IL-1beta, IL-4, IL-10, IL-13, TNF-alpha and IFN-gamma) related to AD can be inhibited, skin inflammatory response is reduced, and meanwhile, the oxidative stress resistance of serum is improved; excessive proliferation of pathogenic bacteria (such as staphylococcus aureus) is inhibited, growth of beneficial bacteria is promoted, and the diversity of skin microorganisms is increased; by enhancing the skin barrier function, water loss and permeation of external irritants are reduced. Based on the pathologic mechanism of AD and the limitation of the existing treatment method, the invention provides an innovative application of relieving AD symptoms and regulating skin microbiota through natural chemical components. The invention has significant technical advantages and application prospects, and provides a new idea and solution for AD treatment.

Description

TECHNICAL FIELD

[0001] The application relates to a new application of a compound, in particular to an application of linalool in preparation of a medicament for treating atopic dermatitis. BACKGROUND

[0002] Atopic dermatitis (AD) is a common chronic inflammatory skin disease. The main pathological mechanisms of AD include: skin barrier defect: due to Filaggrin gene mutation or other factors, the skin barrier function is impaired, the water loss is increased, and the external irritants and allergens are easy to penetrate. Immune system disorder: Th2 type immune response is excessively activated, leading to the release of a large amount of inflammatory factors such as IL-4, IL-13 and IL-31, causing itching and inflammation. Microbiota imbalance: the skin surface microbiota (such as excessive proliferation of Staphylococcus aureus) of AD patients is closely related to the severity of the disease.

[0003] At present, the treatment methods of AD mainly include local treatment, systemic treatment and biological agents, but these methods still have certain limitations, such as glucocorticoids and calcineurin inhibitors, which can relieve symptoms but may cause skin atrophy, telangiectasia and other side effects when used for a long time; immunosuppressive agents such as cyclosporine and methotrexate may cause systemic side effects (such as nephrotoxicity and hepatotoxicity); biological agents such as Dupilumab (anti-IL-4 / IL-13 monoclonal antibody) have certain efficacy, but are expensive and only target specific inflammatory pathways; the existing treatment methods do not fully target the imbalance of skin microbiota, and the microbiota plays an important role in the pathogenesis of AD.

[0004] In recent years, studies have shown that skin microbiota plays a key role in the onset and progression of AD, and the colonization rate of Staphylococcus aureus on the skin surface of AD patients is significantly increased, and the toxins (such as delta-toxin) secreted by Staphylococcus aureus can exacerbate inflammatory reactions. At the same time, the skin microbial diversity of AD patients is significantly reduced, and the abundance of probiotics (such as coagulase-negative staphylococci, lactobacilli and propionibacterium acnes) is reduced. Microbial-immune interaction also plays an important role in the process of the disease, and skin microbiota affects the pathological process of AD by regulating immune response, such as Staphylococcus aureus which can activate Th2 type immune response.

[0005] Some chemical components have been studied for AD treatment, but their mechanisms of action and efficacy still need to be further optimized. Natural active molecules such as glycyrrhizic acid and green tea extract (EGCG) have anti-inflammatory effects but limited effect. Silver nanoparticles and chlorhexidine can inhibit Staphylococcus aureus but may disrupt the balance of microbiota, while prebiotics and postbiotics can regulate microbiota but lack specificity. SUMMARY

[0006] The present application aims to address the following major shortcomings of existing treatments for alleviating AD (atopic dermatitis) symptoms:

[0007] Limited therapeutic effect: Existing treatments (such as topical hormones, immunosuppressants, etc.) can alleviate symptoms, but long-term use may cause skin atrophy, drug resistance, and other problems, and cannot fundamentally solve the pathological mechanism of AD.

[0008] Obvious side effects: Many drugs (such as systemic hormones or immunosuppressants) can cause systemic side effects such as immunosuppression, metabolic disorders, etc., affecting the overall health of patients.

[0009] Unable to regulate skin microbiota: Existing treatments usually do not intervene in the imbalance of skin surface microbiota, while the skin microbiota of AD patients (such as over-proliferation of Staphylococcus aureus) is closely related to disease progression.

[0010] Lack of specificity: Existing drugs mainly work through broad-spectrum anti-inflammatory or immunosuppression, lacking precise regulation of AD-specific pathological mechanisms (such as inflammatory mediators, skin barrier dysfunction).

[0011] Unable to shorten the duration of the disease: Existing treatments mainly focus on symptom relief and cannot effectively shorten the course of AD or prevent recurrence.

[0012] Poor patient compliance: Due to the side effects of long-term medication and poor efficacy, patient compliance is low, affecting treatment effectiveness.

[0013] The application of linalool in the preparation of a drug for treating atopic dermatitis is provided. Research has found that natural chemical component linalool can alleviate AD symptoms and regulate skin microbiota through the following mechanisms: it can inhibit key inflammatory mediators related to AD (such as IgE, IL-1β, IL-4, IL-10, IL-13, TNF-α, IFN-γ), reduce skin inflammatory response, and increase serum antioxidant stress capacity; it can inhibit the over-proliferation of pathogenic bacteria (such as Staphylococcus aureus), promote the growth of beneficial bacteria, and increase skin microbial diversity; and it can enhance skin barrier function, reduce water loss, and reduce the penetration of external irritants.

[0014] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0015] In a first aspect, the present application provides the use of linalool in the preparation of a drug for treating atopic dermatitis.

[0016] Further, linalool inhibits the expression of inflammatory mediators related to atopic dermatitis.

[0017] Further, the inflammation mediators related to atopic dermatitis are IgE, IL-1β, IL-4, IL-10, IL-13, TNF-α and IFN-γ.

[0018] Further, linalool improves the serum antioxidant stress capacity.

[0019] In the second aspect, the present application provides the use of linalool in the preparation of a medicament for regulating skin microbial flora.

[0020] Further, linalool inhibits the excessive proliferation of pathogenic bacteria, promotes the growth of beneficial bacteria, and increases the diversity of skin microorganisms.

[0021] Further, linalool enhances the skin barrier function, reduces water loss and penetration of external irritants.

[0022] In the third aspect, the present application provides the use of a pharmaceutical composition in the preparation of a medicament for treating atopic dermatitis or regulating skin microbial flora, wherein the pharmaceutical composition comprises a therapeutically effective amount of linalool and a pharmaceutically acceptable excipient.

[0023] Further, the pharmaceutically acceptable excipient comprises at least one of a diluent, an excipient, a filler, a binder, a humectant, an absorption enhancer, a surfactant, a lubricant, a stabilizer, a flavoring agent, a sweetener, and a pigment.

[0024] Further, the pharmaceutical composition is a liquid preparation, a solid preparation or a spray preparation.

[0025] In summary, due to the adoption of the above technical solutions, the present application has the following advantages:

[0026] 1. The present application provides the use of linalool in the preparation of a medicament for treating atopic dermatitis. Research has found that natural chemical component linalool can relieve AD symptoms and regulate skin microbial flora through the following mechanisms: it can inhibit key inflammation mediators related to AD (such as IgE, IL-1β, IL-4, IL-10, IL-13, TNF-α, IFN-γ), reduce skin inflammatory response, and improve serum antioxidant stress capacity; it can inhibit the excessive proliferation of pathogenic bacteria (such as Staphylococcus aureus), promote the growth of beneficial bacteria, and increase the diversity of skin microorganisms; and it can enhance the skin barrier function, reduce water loss and penetration of external irritants. Based on the pathological mechanism of AD and the limitations of existing treatment methods, the present application proposes an innovative application of natural chemical components to relieve AD symptoms and regulate skin microbial flora. The present application has significant technical advantages and application prospects, and provides a new idea and solution for AD treatment.

[0027] 2. The application provides application of the aromatic alcohol in preparation of a medicine for treating atopic dermatitis. Research finds that linalool can realize a synergistic treatment effect by simultaneously inhibiting inflammation and regulating microbial flora. The chemical component belongs to a natural plant extract, has high efficiency and low toxicity, and is suitable for long-term use. Moreover, the natural plant component has a wide source and a lower price. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 1 is a diagram of pathological changes of mouse skin.

[0029] Figure 2 Figure 3 is a diagram of statistical data of mouse weight and scratching times.

[0030] Figure 3 Figure 5 is a diagram of data of blood cell changes of mice.

[0031] Figure 4 Figure 7 is a diagram of data of changes of mouse cytokine levels.

[0032] Figure 5 Figure 9 is a diagram of data of changes of serum biochemical indexes.

[0033] Figure 6 Figure 11 is a diagram of data of microbial composition analysis of mouse skin.

[0034] Figure 7 Figure 13 is a diagram of data of successful functional prediction analysis of the microbiome. DETAILED DESCRIPTION

[0035] The application will be described in further detail below with reference to the drawings.

[0036] In order to make the object, technical scheme and advantages of the application more clear, the application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the application and do not limit the application.

[0037] Example 1

[0038] The AD mouse model was constructed by Ovalbumins (OVA). SPF level 6-8 week old C57bl / 6 mice were adaptively fed for one week, and then the first day of systemic sensitization was started. The mice were injected with 200 μg / mL OVA solution 0.5 mL (containing 5% alum adjuvant) intraperitoneally. On the 6th day (systemic sensitization again), the mice were injected with 100 μg / mL OVA solution 0.5 mL subcutaneously on the back. On the 17th day, the mice were anesthetized with sodium barbital, the back hair was removed, and the first round of local sensitization was carried out. After repeatedly stimulating the back skin 8 times with a brush, 1000 μg / mL OVA solution 50 μL was placed on a 0.5 cm2 sterile gauze and attached to the mouse back skin. Fresh OVA solution was replaced daily for one week. At this time, the mice were randomly divided into 5 groups (N=10). The second round of local sensitization (repeat the first round of operation) was started on the 39th day. The model group (AD) was not treated, and the experimental group (5% Linalool) and the positive group (Positive, Pterocarpus marsupium) were treated with the corresponding drugs before OVA. The drug application lasted for two weeks. During the experiment, the mice's food intake and mental state were observed, and the mice's body weight changes were tracked. The blank group (CK) mice were subjected to hair removal without other stimulation.

[0039] (1) Scratching frequency statistics: 1 hour after the last OVA induction, the scratching behavior of the experimental mice was observed. Each time the mice scratched the OVA sensitized skin was counted as 1 effective scratch. Continuous scratching for more than 3 seconds was counted as 2 times, and the mice were manually intervened to stop the scratching behavior after 3 seconds. The number of scratches within 10 minutes was calculated.

[0040] (2) Changes in blood environment: 24 hours after the last sensitization, blood was taken from the mouse heart, serum was separated and collected, and stored at -80°C. The changes in blood cells were detected by a five-class blood cell analyzer. The changes in different indicators were detected by a biochemical analyzer. The concentrations of IgE, IL-4, IL-10, IL-1β, TNF-α, and IFN-γ in serum were detected by biotin double-antibody sandwich enzyme-linked immunosorbent assay according to the kit instructions. The software was used for calculation.

[0041] (3) Antioxidant capacity detection: After the experiment, the serum was used for H2O2 content detection. Colorimetric determination was performed by chemiluminescence method according to the kit instructions, and calculation was performed by software.

[0042] (4) Skin microbial composition: After the experiment, sterile swabs were used to collect physiological saline to collect microbial samples on the skin wound and its surrounding surface of mice. The blank group used sterile swabs to collect microbial samples on the skin of mice, and then suspended them in physiological saline solution. According to the microbial DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.), the sample DNA was extracted, the microbial species distribution was analyzed, and the primer amplification 16S rRNA gene (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT) was used. Microbial composition sequencing and analysis were performed by Shanghai Pinshenino Biotechnology Co., Ltd., and sequencing was performed on Illumina Novaseq. Sequence denoising or OTU clustering was performed according to the analysis process of QIIME2 or Vsearch software. In order to evaluate the diversity level of each sample, the distribution of ASV / OTU in different samples was evaluated, and bioinformatics analysis was performed. Based on the KEGG and COG databases, the differential metabolic pathways were matched using the standardized pathway / group abundance table.

[0043] Skin tissue damage: The skin tissue of the mouse back (OVA sensitized group: allergen contact site skin, non-sensitized group: abdominal skin at the corresponding site) was taken, with a size of about 1.0 cm x 0.5 cm, fixed with 10% formaldehyde solution, and routinely paraffin-embedded, sectioned, and hematoxylin-eosin (HE) stained. The tissue sections were baked in a 65°C oven for about 1.5 h, cooled, and then deparaffinized, then placed in a container containing antigen repair solution and heated to 95°C for 15 min, washed with PBS 3 times for 5 min each time, added rabbit anti-mouse CD4 antibody (1:250), 4°C overnight, washed with PBS 3 times for 5 min each time, then added goat anti-rabbit IgG working solution (1:1000), room temperature, 40 min in the dark. Staining was performed using the streptavidin-peroxidase method, according to the kit instructions, and color development was performed with color developing agent, then filtered paper was used to absorb the color, hematoxylin was re-stained for 30 s, hydrochloric acid ethanol was differentiated, neutral resin was mounted, observed under a microscope, and Image J software was used to analyze and process the pictures.

[0044] Histopathological improvement after linalool treatment

[0045] HE staining showed that the epidermis of the model mice was significantly thickened (170.4 ± 16.35 μm) (p < 0.01), with incomplete keratinization, a small amount or partial degeneration and necrosis of collagen fibers in the dermis, blurred structure of the necrotic collagen fibers, and karyopyknosis and disintegration; accompanied by significant inflammatory cell infiltration and an increase in mast cells compared with the blank control group (p < 0.01). The epidermis of the mice treated with linalool was significantly thinner (64 ± 8.33 μm) (p < 0.01), with increased volume and number of prickle cell layers, accompanied by varying degrees of inflammatory cell infiltration, mainly neutrophilic granulocytes with segmented or rod-shaped nuclei and lymphocytes with oval nuclei, and clear subcutaneous fat layer and muscle layer structure, with orderly arranged adipocytes and muscle fibers and normal nuclear morphology. The number of mast cells was slightly lower than that of the model mice, and the degranulation rate was lower after treatment with linalool. The results are shown in Figure 1 .

[0046] The degree of itching was reduced after treatment with linalool

[0047] The AD mice had obvious itching and frequent scratching (75.86 ± 14.91 times / 30 min), and the body weight decreased rapidly. After treatment with linalool, the scratching was significantly reduced (28.75 ± 5.53 times / 30 min) (p < 0.01), and the body weight increased rapidly, which was comparable to that of the positive control group (32.5 ± 6.82 times / 30 min). The results are shown in Figure 2 .

[0048] The level of inflammation was reduced in the mice treated with linalool

[0049] After the experiment, the blood and serum of the mice in each group were analyzed. Blood cell analysis showed that the white blood cells and lymphocytes of the AD mice decreased (p < 0.01), while the red blood cells and neutrophils increased slightly. After treatment with linalool and the positive drug, the white blood cells and lymphocytes gradually returned to the level of healthy mice, the red blood cells decreased to the level of healthy mice, and the neutrophils did not recover during the observation period Figure 3 . The results of serum ELISA detection are shown in Figure 4 The IgE, IL-10, IL-4, TNF-α, and IFN-γ of the AD mice significantly increased (p < 0.05), and IL-1β also increased slightly, but there was no significant difference. After treatment with linalool, IgE and TNF-α significantly decreased (p < 0.05) (see Figure 4The remaining cytokines also showed a decreasing trend, indicating that the treatment led to a decrease in serum inflammatory factors in mice and suppressed the inflammatory response. Serum biochemical analysis revealed that AD caused significant differences in ALT, AST, CK, CK-MB, and TG levels between AD mice and healthy mice (p < 0.05). After linalool treatment, AST, CK, CK-MB, and TG levels showed significant differences from AD mice and were returning to healthy levels. HDL-C, LDL-C, UREA, and ALB did not show significant differences among AD mice, healthy mice, and linalool-treated mice, but linalool treatment significantly increased HDL-C, LDL-C, and UREA (p < 0.05). Figure 5 In addition, linalool treatment also reduced serum hydrogen peroxide levels in AD mice and enhanced their antioxidant capacity. Figure 2 This indicates that linalool can regulate oxidative stress and biochemical reactions in mice, thus helping to maintain homeostasis.

[0050] Linalool regulates the skin microecology

[0051] Species identification and analysis were performed on the skin microbiota of AD mice, CK mice, and linalool-treated mice. The abundance of characteristic sequences was characterized through steps including primer removal, quality filtering, noise reduction, splicing, chimera removal, and duplicate removal. A total of 23 common microbial species were found among the three groups. The AD group had the most species (1774 species), followed by the CK group (512 species), while the linalool-treated group had the fewest (412 species). Results are shown below. Figure 6 Staphylococcus was the dominant species in the three groups, but its abundance was the lowest in the AD group, and its abundance rebounded after linalool treatment. Escherichia-Shigella, Enterobacter, Neisseria, Leptotrichia, and Glutamicibacter were more abundant in the AD group, but absent or very low in the CK and linalool treatment groups. Pseudomonas, Cutibacterium, Brevundimonas, and Enterorhabdus were more prevalent in the CK and linalool treatment groups, but less so in the AD group. Agrococcus, Microbacterium, Olsenella, and Methylobacterium-Methylorubrum were specific to the CK group. In general, the more abundant species in the AD group were mostly potentially pathogenic harmful species, while the CK and linalool treatment groups had more beneficial species. Figure 6 ABC). Alpha diversity analysis showed no significant differences in species richness, diversity, and evenness among samples within each group. Figure 6D), indicating no difference in within-sample habitat diversity. Beta diversity analysis showed that CK and linalool treatment groups had more similar species composition and diversity, while AD group had a large difference in composition Figure 6 E), clustering analysis confirmed that CK and linalool treatment groups were more similar to each other and far from AD group Figure 6 F, G). Niche analysis also revealed significant differences in microbial niche breadth among the three groups Figure 7 D). Dominant species difference test on the different species found that Staphylococcus was the dominant species between AD and linalool treatment groups, with lower abundance in AD group, which was corrected by linalool Figure 7 A), while random forest analysis after machine learning found that Staphylococcus lentuss, Jeotgalicoccuss, Staphylococcus were the most important three species, all highly expressed in CK and linalool treatment groups and lowly expressed in AD group Figure 6 H). Bugbase phenotype prediction showed that these microorganisms were related to Gram Positive, StressTolerant, Contains Mobile, Elements, Facultatively_Anaerobic, Gram Negative, Anaerobic, Aerobic, and notably, microorganisms in AD group were related to Potentially Pathogenic, Forms Biofilms Figure 7B) The potential functional prediction of the microorganism related to the path of the disease includes chemoheterotrophy, aerobic chemoheterotrophy, nitrate reduction, fermentation, animal parasites or symbionts, human associated, ureolysis, human pathogens all, human gut, mammal gut, respiration of sulfur compounds, dark hydrogen oxidation, aromatic compound degradation, nitrate respiration, nitrogen respiration, sulfate respiration, sulfur respiration, nitrite respiration, denitrification, nitrate denitrification, while the contribution of AD is more fermentation, human gut, mammal gut, while the aerobic chemoheterotrophy, nitrogen respiration is more in the CK and linalool treatment group Figure 7 C) In summary, after linalool treatment of AD mice, the composition of the skin surface microorganism can be improved, the abundance of harmful microorganism is reduced, the abundance of beneficial microorganism is increased, the skin surface microecological balance is maintained, and the AD condition is improved in cooperation with the body inflammation system.

[0052] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of linalool in the preparation of drugs for treating atopic dermatitis.

2. The application according to claim 1, characterized in that, Linalool inhibits the expression of inflammatory mediators associated with atopic dermatitis.

3. The application according to claim 2, characterized in that, The inflammatory mediators associated with atopic dermatitis include IgE, IL-1β, IL-4, IL-10, IL-13, TNF-α, and IFN-γ.

4. The application according to claim 1, characterized in that, Linalool enhances serum antioxidant stress resistance.

5. Application of linalool in the preparation of drugs that regulate skin microbiota.

6. The application according to claim 5, characterized in that, Linalool inhibits the overgrowth of pathogenic bacteria, promotes the growth of beneficial bacteria, and increases the diversity of skin microbiota.

7. The application according to claim 5, characterized in that, Linalool enhances the skin barrier function, reducing moisture loss and the penetration of external irritants.

8. The use of a pharmaceutical composition in the preparation of a drug for treating atopic dermatitis or a drug for regulating skin microbiota, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of linalool, and pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, Pharmaceutically acceptable excipients include at least one of the following: diluents, excipients, fillers, binders, humectants, absorption enhancers, surfactants, lubricants, stabilizers, flavorings, sweeteners, and colorings.

10. The application according to claim 8 or 9, characterized in that, The pharmaceutical composition is a liquid formulation, a solid formulation, or a spray formulation.

Images