Application method of thuja sutchuenensis volatile oil in preparation of products for treating skin diseases

Abstract

The invention belongs to the technical field of skin treatment, discloses an application method of thuja sutchuenensis volatile oil in preparation of products for treating skin diseases, establishes an omnibearing and multi-target treatment strategy of thuja sutchuenensis volatile oil, and provides a very important theoretical support for development of atopic dermatitis resistant products and utilization and conversion of natural products. The treatment strategy can provide a scientific basis for natural therapy and promote application of the natural therapy in clinical practice. According to the present invention, with the combination of the pharmaceutical professional knowledge and the experimental technology, the understanding on phytochemistry and pharmacology is deepened, the scientific basis is provided for the development and the utilization of the plant essential oil, and the research and the development in the medicine field are promoted. The scratching frequency of mice treated by using the thuja sutchuenensis volatile oil is obviously reduced, the treatment process is advanced by one week, and skin injury is recovered quickly; the AD mice suffer from weight loss obviously, and the weight of the mice in the treatment group recovers quickly.

Description

Technical Field

[0001] The invention belongs to the technical field of skin treatment, and in particular relates to an application method of thuja volatile oil in preparing a product for treating skin diseases. Background Art

[0002] Atopic dermatitis (AD) is a common chronic, recurrent, inflammatory skin disease characterized by systemic dryness, itching, erythema, and recurrent skin lesions, which greatly affects the patient's quality of life. Globally, the prevalence of AD has increased year by year, and it is common in children and young adults, with a prevalence of 15-20% in children and 1-3% in adults. Although atopic dermatitis itself is not directly life-threatening, its chronic and recurrent characteristics often put patients under long-term treatment pressure and increase the medical burden. In addition, AD is often associated with asthma and allergic diseases, and AD has become one of the important risk factors due to increased infections and reduced psychosocial and physical quality of life. No single cause of AD has been found. Instead, the pathophysiology of AD seems to involve multiple factors, including host genetics, altered skin barrier function, and immune abnormalities. The corresponding treatment research mainly focuses on immune regulation, skin barrier repair and inhibition of inflammatory response. Therapeutic drugs include steroid drugs, calcineurin inhibitors, etc., but the side effects, drug resistance and recurrence of these drugs are still the main problems in clinical treatment. At present, the treatment of atopic dermatitis is mainly based on topical medications (such as corticosteroids) and immunosuppressants, but long-term use of these drugs may cause a series of side effects, including skin atrophy and immune suppression. In addition, the pathogenesis of atopic dermatitis is complex, involving multiple factors such as the immune system, skin barrier function, and microbial community imbalance. Treatment requires a multi-pronged approach aimed at controlling inflammation (anti-inflammatory), itching (antipruritic), bacterial superinfection (antibacterial) and skin barrier restoration (moisturizer). Recent studies have shown that natural products and Chinese herbal medicines have potential application value in the treatment of atopic dermatitis. Traditional Chinese medicine has a long tradition of treating rash diseases. Due to its low cost, good efficacy and good safety, traditional Chinese medicine, as a complementary and alternative medicine, is of great significance in the development of AD therapeutic drugs. Studies have shown that more and more natural active ingredients can exhibit ideal therapeutic effects by regulating immune responses, improving skin barrier function or alleviating inflammatory responses.

[0003] Through the above analysis, the problems and defects of the prior art are as follows:

[0004] The treatment of atopic dermatitis mainly relies on topical medications (such as corticosteroids) and immunosuppressants, but long-term use of these drugs may cause a series of side effects, including skin atrophy, immune suppression, etc. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for applying Thuja sutchuenensis volatile oil in preparing a product for treating skin diseases.

[0006] The present invention is achieved in that a method for applying Thuja sutchuenensis volatile oil in preparing a product for treating skin diseases comprises:

[0007] Step 1, counting the number of scratches;

[0008] Step 2, changes in blood environment;

[0009] Step 3, antioxidant capacity detection;

[0010] Step 4, skin microbial composition;

[0011] Step 5, skin tissue damage.

[0012] Further, the scratching times are counted:

[0013] The scratching behavior of the experimental mice was observed 1 hour after the last ovalbumin (OVA) induction. Scratching the skin of the OVA-sensitized area was counted as one effective scratch, and continuous scratching for more than 3 seconds was counted as two scratches. After 3 seconds, the mice were manually intervened to stop the scratching behavior, and the number of scratches within 10 minutes was calculated.

[0014] Furthermore, the blood environment changes:

[0015] 24 hours after the last sensitization, blood was collected from the mouse heart, and serum was separated and collected and stored at -80℃; whole blood was tested for blood cell changes using a five-category blood cell analyzer; serum was tested for changes in different indicators using a biochemical analyzer; the concentrations of immunoglobulin E (Immunoglobulin E, IgE), interleukin-4 (Interleukin-4, IL-4), interleukin-10 (Interleukin-10, IL-10), interleukin-1beta (Interleukin-1beta, IL-1β), tumor necrosis factor alpha (Tumor Necrosis Factoralpha, TNF-α), and interferon gamma (Interferon gamma, IFN-γ) in serum were measured using biotin double antibody sandwich enzyme-linked immunosorbent assay, which was performed according to the instructions of the kit and calculated using software.

[0016] Further, the antioxidant capacity is detected:

[0017] After the test, the serum was used for H 2 O 2The total iron binding capacity was detected by chemiluminescence colorimetry according to the kit instructions and calculated by software.

[0018] Furthermore, the skin microbial composition is:

[0019] After the experiment, sterile swabs were dipped in saline to collect microbial samples from the mouse skin wounds and their surrounding surfaces. In the blank group, sterile swabs were used to collect microbial samples from the mouse skin, which were then suspended in saline solution. According to the instructions of the Microbial DNA Extraction Kit (Tiangen Biotechnology (Beijing) Co., Ltd.), sample DNA was extracted, the distribution of microbial species was analyzed, and the 16S rRNA gene was amplified using primers (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT). Microbial composition sequencing and analysis were performed by Shanghai Pisenuo Biotechnology Co., Ltd. and sequenced 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, standardized pathway / group abundance tables were used to match differential metabolic pathways.

[0020] Further, the skin tissue damage:

[0021] The skin tissues of the back of mice (the skin of the allergen contact site in the OVA-sensitized group and the abdominal skin of the corresponding site in the non-sensitized group) were obtained, with a size of about 1.0 cm × 0.5 cm, and fixed with 10% formaldehyde solution, routinely paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE), observed under a microscope, and the images were analyzed and processed using Image J software.

[0022] Another object of the present invention is to provide an application system of Thuja sutchuenensis volatile oil in preparing a product for treating skin diseases, comprising:

[0023] The scratching frequency statistics module is used to observe the scratching behavior of experimental mice 1 hour after the last OVA induction. The scratching of the skin of the OVA-sensitized area by the mouse is counted as one effective scratch, and continuous scratching for more than 3 seconds is counted as two times. After 3 seconds, the mouse is manually intervened to stop the scratching behavior, and the scratching frequency within 10 minutes is calculated;

[0024] The blood environment change module is used to collect blood from the mouse heart 24 hours after the last sensitization, separate and collect serum, and store it at -80℃; the whole blood is tested for blood cell changes by a five-category blood cell analyzer; the serum is tested for changes in different indicators by a biochemical analyzer; the concentrations of IgE, IL-4, IL-10, IL-1β, TNF-α, and IFN-γ in the serum are measured by biotin double antibody sandwich enzyme-linked immunosorbent assay, which is operated according to the instructions of the kit and calculated using software;

[0025] Antioxidant capacity detection module, used for serum after the test for H 2 O 2 and total iron binding capacity detection, colorimetric determination was performed using chemiluminescence according to the kit instructions and calculated using software;

[0026] Skin microbial composition module, used to collect microbial samples from mouse skin wounds and their surrounding surfaces with sterile swabs dipped in saline after the experiment, and the blank group used sterile swabs to collect microbial samples from mouse skin, which were then suspended in saline solution; according to the instructions of the microbial DNA extraction kit (Tiangen Biotechnology (Beijing) Co., Ltd.), sample DNA was extracted, microbial species distribution was analyzed, and 16S rRNA gene was amplified using primers (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT); microbial composition sequencing and analysis were performed by Shanghai Pisenuo Biotechnology Co., Ltd. and sequenced 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, standardized pathway / group abundance tables were used to match differential metabolic pathways;

[0027] The skin tissue injury module was used to obtain skin tissue from the back of mice (the skin of the allergen contact site was obtained in the OVA-sensitized group, and the abdominal skin of the corresponding site was obtained in the non-sensitized group). The size was about 1.0 cm × 0.5 cm. The skin tissue was fixed with 10% formaldehyde solution, routinely paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE); observed under a microscope, and the images were analyzed and processed using Image J software.

[0028] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0029] First, in view of the technical problems existing in the above-mentioned prior art, some creative technical effects are brought about after solving the problems. The specific description is as follows:

[0030] 1. Provide new ideas and methods for the treatment of inflammatory diseases; its ingredients may contain compounds that are effective in treating inflammation-related diseases, such as adjuvant cancer treatment, arthritis, chronic diseases, etc.

[0031] 2. Inflammatory diseases, as concomitant diseases, are closely related to human health. Many traditional herbs and plant extracts are used to treat inflammatory diseases. Establishing a "comprehensive, multi-target" treatment strategy for Thuja sutchuenensis volatile oil provides very important theoretical support for the development of anti-atopic dermatitis products and the utilization and transformation of natural products. This treatment strategy can provide a scientific basis for natural therapies and promote their application in clinical practice. Combining pharmaceutical expertise and experimental techniques, we can deepen our understanding of plant chemistry and pharmacology, provide a scientific basis for the development and utilization of plant essential oils, and promote research and development in the medical field.

[0032] The number of scratching times of mice treated with Thuja sutchuenensis volatile oil was significantly reduced, the treatment process was advanced by one week, and skin damage recovered faster; AD mice lost a significant amount of weight, and mice in the treatment group recovered their weight faster.

[0033] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0034] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0035] Through market research and analysis of skin disease treatment needs, it was found that compared with traditional hormone drugs, Thuja essential oil as a natural product has a unique affinity in the market. In particular, its characteristics as a natural product meet the application prospects in treatment needs from the aspects of high safety and few side effects. As a new treatment option, Thuja essential oil may have broad market prospects.

[0036] (2) Whether the technical solution of the present invention solves the technical problem that people have been eager to solve but have never been able to solve successfully: the treatment of atopic dermatitis is mainly based on topical medications (such as corticosteroids) and immunosuppressants, but long-term use of these drugs may cause a series of side effects (skin atrophy, immune suppression, etc.). The present invention also demonstrates how Thuja essential oil can effectively overcome these problems as a new treatment option and provide better therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The present invention provides a flowchart of a method for applying Thuja sutchuenensis volatile oil in preparing a product for treating skin diseases.

[0038] Figure 2 It is a system structure diagram of the application method of Thuja sutchuenensis volatile oil in preparing products for treating skin diseases provided by an embodiment of the present invention.

[0039] Figure 3 The physical signs of AD mice treated with Thuja sutchuenensis volatile oil provided in the embodiment of the present invention are shown in Table 1. A, skin changes of AD mice, CK, saline group, 30Day, 30 days after OVA modeling, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis volatile oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis volatile oil treatment after modeling; B, weight changes of AD mice during the observation period (1-5 weeks), CK, saline group, Model, OVA modeling Model; Positive, group treated with putepic tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling; C, statistics of the number of scratching times of mice after administration (4th and 5th weeks), Model, OVA model, Positive, group treated with putepic tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling.

[0040] Figure 4 The blood cell changes of AD mice provided in the embodiment of the present invention; CK, saline group, Model, OVA modeling; Positive, group treated with tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0041] Figure 5 The changes in biochemical indicators of AD mice provided in the embodiments of the present invention; CK, saline group, Model, OVA modeling; 5% YB, 5% Thuja volatile oil treatment after modeling, 10% YB, 10% Thuja volatile oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0042] Figure 6 The volatile oil of Thuja juniper provided in the embodiment of the present invention regulates the level of inflammatory factors; CK, saline group, Model, OVA modeling; 5% YB, 5% Thuja juniper volatile oil treatment after modeling, 10% YB, 10% Thuja juniper volatile oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0043] Figure 7 The serum antioxidant capacity level of AD mice treated with Thuja sutchuenensis volatile oil provided in the embodiment of the present invention; CK, saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis volatile oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis volatile oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0044] Figure 8 The AD mouse pathological tissue changes provided by the embodiment of the present invention; CK, saline group, Model, OVA modeling; 5% YB, 5% Thuja volatile oil treatment after modeling, 10% YB, 10% Thuja volatile oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0045] Fig. 9 The following are the changes in the skin microbial composition of AD mice provided in the embodiments of the present invention; A, species composition analysis, B, species composition Sankey diagram, C, species composition evolution tree analysis, D, α diversity analysis, E, niche analysis. CK, saline group, Model, OVA modeling; YB, treated with 5% Thuja volatile oil after modeling. *p≤0.05, p≤0.01. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] like Figure 1 As shown, the application method of Thuja sutchuenensis volatile oil in preparing a product for treating skin diseases provided by an embodiment of the present invention comprises the following steps:

[0048] S101, scratching count;

[0049] S102, changes in blood environment;

[0050] S103, antioxidant capacity test;

[0051] S104, skin microbiome composition;

[0052] S105, skin tissue damage.

[0053] The scratching frequency statistics provided by the embodiment of the present invention are as follows:

[0054] The scratching behavior of the experimental mice was observed 1 hour after the last OVA induction. Scratching the skin of the OVA-sensitized area was counted as one effective scratch, and continuous scratching for more than 3 seconds was counted as two scratches. After 3 seconds, the mice were manually intervened to stop the scratching behavior, and the number of scratches within 10 minutes was calculated.

[0055] The blood environment changes provided by the embodiments of the present invention are:

[0056] 24 hours after the last sensitization, blood was collected from the mouse heart, and serum was separated and collected and stored at -80℃; whole blood was tested for blood cell changes using a five-category blood cell analyzer; serum was tested for changes in different indicators using a biochemical analyzer; serum IgE, IL-4, IL-10, IL-1β, TNF-α, and IFN-γ concentrations were measured using biotin double antibody sandwich enzyme-linked immunosorbent assay, which was performed according to the kit instructions and calculated using software.

[0057] Antioxidant capacity detection provided by the embodiment of the present invention:

[0058] After the test, the serum was used for H 2 O 2 The total iron binding capacity was detected by chemiluminescence colorimetry according to the kit instructions and calculated by software.

[0059] The skin microbial composition provided by the embodiment of the present invention is:

[0060] After the experiment, sterile swabs were dipped in saline to collect microbial samples from the mouse skin wounds and their surrounding surfaces. In the blank group, sterile swabs were used to collect microbial samples from the mouse skin, which were then suspended in saline solution. According to the instructions of the Microbial DNA Extraction Kit (Tiangen Biotechnology (Beijing) Co., Ltd.), sample DNA was extracted, the distribution of microbial species was analyzed, and the 16S rRNA gene was amplified using primers (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT). Microbial composition sequencing and analysis were performed by Shanghai Pisenuo Biotechnology Co., Ltd. and sequenced 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, standardized pathway / group abundance tables were used to match differential metabolic pathways.

[0061] Skin tissue damage provided by the embodiments of the present invention:

[0062] The skin tissues of the back of mice (the skin of the allergen contact site in the OVA-sensitized group and the abdominal skin of the corresponding site in the non-sensitized group) were obtained, with a size of about 1.0 cm × 0.5 cm, and fixed with 10% formaldehyde solution, routinely paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE); observed under a microscope, and the images were analyzed and processed using Image J software.

[0063] like Figure 2As shown, an application system of Thuja sutchuenensis volatile oil in preparing a product for treating skin diseases provided by an embodiment of the present invention comprises:

[0064] The scratching frequency statistics module is used to observe the scratching behavior of experimental mice 1 hour after the last OVA induction. The scratching of the skin of the OVA-sensitized area by the mouse is counted as one effective scratch, and continuous scratching for more than 3 seconds is counted as two times. After 3 seconds, the mouse is manually intervened to stop the scratching behavior, and the scratching frequency within 10 minutes is calculated;

[0065] The blood environment change module is used to collect blood from the mouse heart 24 hours after the last sensitization, separate and collect serum, and store it at -80℃; the whole blood is tested for blood cell changes by a five-category blood cell analyzer; the serum is tested for changes in different indicators by a biochemical analyzer; the concentrations of IgE, IL-4, IL-10, IL-1β, TNF-α, and IFN-γ in the serum are measured by biotin double antibody sandwich enzyme-linked immunosorbent assay, which is operated according to the instructions of the kit and calculated using software;

[0066] Antioxidant capacity detection module: After the test, serum is used for H2O2 and total iron binding capacity detection. The chemiluminescence method is used for colorimetric determination according to the kit instructions, and the calculation is performed by software;

[0067] Skin microbial composition module, used to collect microbial samples from mouse skin wounds and their surrounding surfaces with sterile swabs dipped in saline after the experiment, and the blank group used sterile swabs to collect microbial samples from mouse skin, which were then suspended in saline solution; according to the instructions of the microbial DNA extraction kit (Tiangen Biotechnology (Beijing) Co., Ltd.), sample DNA was extracted, microbial species distribution was analyzed, and 16S rRNA gene was amplified using primers (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT); microbial composition sequencing and analysis were performed by Shanghai Pisenuo Biotechnology Co., Ltd. and sequenced 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, standardized pathway / group abundance tables were used to match differential metabolic pathways;

[0068] The skin tissue injury module was used to obtain skin tissue from the back of mice (the skin of the allergen contact site was obtained in the OVA-sensitized group, and the abdominal skin of the corresponding site was obtained in the non-sensitized group), with a size of about 1.0 cm × 0.5 cm, fixed with 10% formaldehyde solution, routinely paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE); the images were analyzed and processed using Image J software.

[0069] After the volatile oil of Thuja sutchuenensis is applied to the affected area in the form of an external product (such as an ointment or spray), it first acts on the nerve endings through the active ingredients, regulates the skin sensory signals, and relieves the itching symptoms. The scratching behavior is recorded using a specific animal model (such as a mouse model), and the inhibitory effect of the volatile oil of Thuja sutchuenensis on skin itching is evaluated by an automatic scratching recording device or manual counting of the number of scratches. The significant difference in the number of scratches between the control group and the experimental group can directly reflect the anti-itching effect of the volatile oil.

[0070] The active ingredients of Thuja sutchuenensis essential oil can be absorbed through the skin into the local microcirculation, reducing the release of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The experiment collected blood samples and detected changes in the concentration of inflammatory factors to evaluate its anti-inflammatory effect. In addition, changes in the levels of oxidative stress markers (such as malondialdehyde (MDA) and glutathione (GSH)) in the blood also reflect the regulatory effect of essential oils on systemic oxidative balance.

[0071] The volatile oil of Thuja sutchuenensis contains a large amount of natural antioxidant components (such as monoterpenoids and phenolic substances), which can enhance the antioxidant capacity of the skin by scavenging free radicals and inhibiting lipid peroxidation. The antioxidant effect of volatile oils can be quantitatively evaluated by using chemical analysis methods (such as DPPH free radical scavenging experiment or FRAP antioxidant capacity test). The results show that the antioxidant effect of volatile oils in damaged skin areas can significantly reduce oxidative damage caused by ultraviolet rays or other external factors.

[0072] The antibacterial properties of Thuja sutchuenensis essential oil can effectively regulate the balance of skin microecology, inhibit the growth of pathogens (such as Staphylococcus aureus), promote the reproduction of beneficial bacteria (such as Lactobacillus), and restore the skin barrier function. The regulatory effect can be confirmed by analyzing the changes in the skin microbiome through 16S rRNA sequencing technology. At the same time, the repair of skin tissue was observed through pathological sections, and the changes in epidermal thickness, dermal collagen fiber arrangement and inflammatory cell infiltration were recorded, further confirming the therapeutic effect of essential oil on skin diseases.

[0073] This method comprehensively evaluates the mechanism and efficacy of Thuja sutchuenensis volatile oil in the treatment of skin diseases by comprehensively analyzing the scratching frequency, blood indexes, antioxidant capacity, microbial composition and tissue repair data.

[0074] 1. Changes in physical signs of AD mice

[0075] Through the adjustment of different sensitizers and different sensitization cycles, a procedure for the stable preparation of AD models has been obtained, that is, after OVA systemic sensitization (2 rounds) and local sensitization (2 rounds), a stable and highly reproducible AD model can be obtained ( Figure 3 A), from the appearance, it can be seen that during the sensitization process, mice will experience rash, cracking, and scabs before medication. The weight tracking results of mice showed that weight loss occurred in the 2nd and 3rd weeks after sensitization; from the 4th week, the weight of mice began to rise. It can be seen that the weight of mice in the YB group increased more ( Figure 3 B). According to the statistics of the number of mouse paw scratching, the number of positive drug paw scratching was the least during the statistical period, followed by the linalool group, which was lower than the model group in the 4th and 5th weeks respectively ( Figure 3 C).

[0076] Figure 3 Effect of Thuja sutchuenensis essential oil on the improvement of physical signs in AD mice

[0077] Note: A, skin changes of AD mice, CK, saline group, 30Day, 30 days after OVA modeling, Model, OVA modeling; 5% YB, treated with 5% thuja volatile oil after modeling, 10% YB, treated with 10% thuja volatile oil after modeling; B, body weight changes of AD mice during the observation period (1-5 weeks), CK, saline group, Model, OVA modeling; Positive, group treated with putibic tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling; C, statistics of the number of scratching of mice after administration (4th and 5th weeks), Model, OVA modeling, Positive, group treated with putibic tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling.

[0078] Figure 4 Changes in blood cells in AD mice

[0079] Note: CK, normal saline group, Model, OVA modeling; Positive, group treated with tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0080] 2. Changes in physiological and biochemical indicators of AD mice

[0081] The results of blood cell analysis after treatment of AD mice showed that after treatment with positive drugs and Thuja volatile oil, the WBC, Lym# and RBC of mice recovered compared with the model group, but did not return to the level of healthy mice, indicating that the treatment may have a beneficial effect on the immune system and hematopoietic system of mice, which helps to improve the body's immune defense ability and blood function, thereby improving overall health. These improvements provide physiological support for the treatment effect, indicating that the treatment may have a restorative effect by improving immune response and hematopoietic function. Similar situations were also observed in biochemical indicators, among which ALT, TG, HDL-C, AST, CK, and CK-MB recovered compared with the model group, but UREA, LDH, and LDL-C were not alleviated, improving the function of the liver and heart, reducing the damage or inflammation of related organs; promoting the normalization of lipid metabolism, and reducing the risk of cardiovascular disease. Overall, the treatment helps to restore normal physiological functions, which may work through mechanisms such as reducing oxidative stress, anti-inflammatory or improving organ repair. These improvements provide physiological support for the treatment effect, indicating that the treatment may have a beneficial effect on the body through multiple mechanisms.

[0082] Figure 5 Changes of biochemical indicators in AD mice

[0083] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0084] 3. Anti-inflammatory activity of Thuja sutchuenensis volatile oil

[0085] The levels of related inflammatory factors in the serum of mice treated with Thuja sutchuenensis volatile oil were detected. Figure 6 , Thuja volatile oil and positive drugs can significantly reduce the serum levels of IL-4, TNF-α, IFN-γ, and IgE (p≤0.05), and there is a decreasing trend for IL-10 and IL-1β, but it did not reach a significant level (p>0.05), and reached a level equivalent to that of the positive drug group, indicating that Thuja volatile oil has the effect of inhibiting inflammatory factors and may control the course of AD by reducing inflammatory responses.

[0086] Figure 6 Thuja volatile oil regulates the level of inflammatory factors

[0087] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0088] 4. Evaluation of the antioxidant capacity of Thuja sutchuenensis volatile oil

[0089] After Thuja volatile oil was used to treat AD mice, serum H2O2 concentration and total iron binding capacity were significantly increased. Figure 7 , Thuja volatile oil and positive drugs can reduce the level of AD mice after treatment, but 5% Thuja volatile oil treatment has the most significant effect (p ≤ 0.05), while 10% Thuja volatile oil has the same effect as positive drugs.

[0090] Figure 7 Serum antioxidant capacity level of Thuja sutchuenensis essential oil in the treatment of AD mice

[0091] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0092] 5. Alleviation of pathological state of AD mice

[0093] The mice in different treatment groups were subjected to histopathological sectioning and HE and toluidine blue staining. Figure 8 HE staining showed that the epidermal structure of the skin tissue was intact and clear. The epidermis of the mice in the model group was thickened, and the volume and number of prickle cells increased, which was significantly different from the control group and the 5% thuja treatment group (p≤0.05). A small amount or part of the collagen fibers in the dermis were degenerated and necrotic, the structure of the necrotic collagen fibers was blurred, and the nuclei were condensed and collapsed; the dermis was accompanied by varying degrees of inflammatory cell infiltration, mainly neutrophils with lobed nuclei or rod-shaped nuclei and lymphocytes with oval nuclei, while in the 5% thuja volatile oil treatment group, a small amount or part of the fibrous tissue in the dermis proliferated, the number of fibroblasts with oblong nuclei increased, and the arrangement of the proliferating fibroblasts was more disorderly. The subcutaneous fat layer and muscle layer structure were clearly visible, the fat cells and muscle fibers were neatly arranged, and the cell nuclear morphology was normal. Toluidine blue staining showed mast cells and degranulated mast cells. The model group had the most mast cells, which was significantly different from the control group (p≤0.01). After treatment with 5% Thuja sutchuenensis essential oil, the number of mast cells decreased (p>0.05), but did not return to a healthy level.

[0094] Figure 8 Pathological changes of AD mice

[0095] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0096] 6. Changes in skin microbial homeostasis in AD mice

[0097] The skin microorganisms of AD mice were sequenced and identified, and the changes in their microbial composition under different treatments were analyzed. Fig. 9 . The species composition showed differences between groups ( Fig. 9 A), Staphylococcus lentus was the dominant species in the Thuja volatile oil and blank groups, while Arthrobacter sp. was the dominant species in the model group; the overlap of species between the blank group and the Thuja volatile oil treatment group was high ( Fig. 9 B, C), the model group had more bacterial species and lower relative abundance, but the species composition diversity among the three groups was not significantly different ( Fig. 9 D), from the analysis of the ecological niche occupied by the bacterial species composition, the ecological niches among the three groups were significantly different ( Fig. 9 E). It can be seen that the skin microbial balance of AD mice was destroyed, and the bacterial species were rich, but the composition of skin microorganisms was quite different from that of healthy mice in the blank group. The mice treated with Thuja sutchuenensis volatile oil showed similar composition and abundance of skin microorganisms to those of healthy mice. Therefore, Thuja sutchuenensis volatile oil may play a therapeutic effect in treating AD by regulating the composition of skin microorganisms and maintaining the balance of skin microorganisms.

[0098] Fig. 9 Changes in skin microbial composition of AD mice; Note: A, species composition analysis, B, species composition Sankey diagram, C, species composition evolution tree analysis, D, α diversity analysis, E, niche analysis. CK, saline group, Model, OVA modeling; YB, treated with 5% Thuja volatile oil after modeling. *p≤0.05, p≤0.01.

[0099] Embodiment 1: preparation and application of thuja volatile oil ointment

[0100] 1. Preparation of ointment:

[0101] Materials: Thuja volatile oil, vaseline, lanolin and liquid paraffin.

[0102] Ratio: Thuja volatile oil accounts for 2%, Vaseline 40%, lanolin 30%, and liquid paraffin 28%.

[0103] step:

[0104] 1. Heat Vaseline and lanolin to 60℃ and stir well to form a matrix.

[0105] 2. When the matrix cools to 40°C, add Thuja volatile oil and liquid paraffin, and continue stirring until completely mixed.

[0106] 3. Divide the ointment into sealed containers and store at room temperature for later use.

[0107] 2. Application:

[0108] Animal model: An OVA-sensitized mouse model was established to induce skin allergic inflammation.

[0109] Dosage: Apply the ointment evenly to the allergic area on the back of the mouse, twice a day for 7 days.

[0110] Effect observation: After ointment treatment, the number of scratching times of mice was significantly reduced, the levels of skin inflammatory factors (such as IgE and TNF-α) were reduced, and HE staining showed obvious skin tissue repair.

[0111] Embodiment 2: Preparation and application of Thuja sutchuenensis volatile oil spray

[0112] 1. Spray preparation:

[0113] Materials: Thuja sutchuenensis essential oil, ethanol, glycerin and purified water.

[0114] Ratio: 1% Thuja volatile oil, 10% ethanol, 5% glycerol, 84% purified water.

[0115] step:

[0116] 1. Mix the volatile oil of Thuja sutchuenensis with ethanol, stir well and then slowly add glycerin.

[0117] 2. Gradually add purified water while stirring, mix well and filter to ensure that the solution is free of particulate impurities.

[0118] 3.Put the prepared solution into a spray bottle and store it at low temperature and away from light.

[0119] 2. Application:

[0120] Animal model: A mouse skin microbial disorder model was used to simulate eczema-like skin lesions.

[0121] Dosage: Spray Thuja sutchuenensis volatile oil spray on the damaged area of ​​the mouse back skin 3 times a day for 10 consecutive days.

[0122] Effect observation: After treatment, the microbial diversity of mouse skin was significantly restored, inflammatory symptoms were alleviated, antioxidant capacity was enhanced, and 16S rRNA gene sequencing showed that the beneficial microbial population increased significantly.

[0123] 2. Relevant evidence of the technical effects obtained by the embodiments of the present invention.

[0124] 1. Changes in physical signs of AD mice

[0125] Through the adjustment of different sensitizers and different sensitization cycles, a procedure for the stable preparation of AD models has been obtained, that is, after OVA systemic sensitization (2 rounds) and local sensitization (2 rounds), a stable and highly reproducible AD model can be obtained ( Figure 3A), from the appearance, it can be seen that during the sensitization process, mice will experience rash, cracking, and scabs before medication. The weight tracking results of mice showed that weight loss occurred in the 2nd and 3rd weeks after sensitization; from the 4th week, the weight of mice began to rise. It can be seen that the weight of mice in the YB group increased more ( Figure 3 B). According to the statistics of the number of mouse paw scratching, the number of positive drug paw scratching was the least during the statistical period, followed by the linalool group, which was lower than the model group in the 4th and 5th weeks respectively ( Figure 3 C).

[0126] Figure 3 Effect of Thuja sutchuenensis essential oil on the improvement of physical signs in AD mice

[0127] Note: A, skin changes of AD mice, CK, saline group, 30Day, 30 days after OVA modeling, Model, OVA modeling; 5% YB, treated with 5% thuja volatile oil after modeling, 10% YB, treated with 10% thuja volatile oil after modeling; B, body weight changes of AD mice during the observation period (1-5 weeks), CK, saline group, Model, OVA modeling; Positive, group treated with putibic tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling; C, statistics of the number of scratching of mice after administration (4th and 5th weeks), Model, OVA modeling, Positive, group treated with putibic tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling.

[0128] Figure 4 Changes in blood cells in AD mice

[0129] Note: CK, normal saline group, Model, OVA modeling; Positive, group treated with tacrolimus after modeling, 5% YB, group treated with 5% thuja volatile oil after modeling, 10% YB, group treated with 10% thuja volatile oil after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0130] 2. Changes in physiological and biochemical indicators of AD mice

[0131] The results of blood cell analysis after treatment of AD mice showed that after treatment with positive drugs and Thuja volatile oil, the WBC, Lym# and RBC of mice recovered compared with the model group, but did not return to the level of healthy mice, indicating that the treatment may have a beneficial effect on the immune system and hematopoietic system of mice, which helps to improve the body's immune defense ability and blood function, thereby improving overall health. These improvements provide physiological support for the treatment effect, indicating that the treatment may have a restorative effect by improving immune response and hematopoietic function. Similar situations were also observed in biochemical indicators, among which ALT, TG, HDL-C, AST, CK, and CK-MB recovered compared with the model group, but UREA, LDH, and LDL-C were not alleviated, improving the function of the liver and heart, reducing the damage or inflammation of related organs; promoting the normalization of lipid metabolism, and reducing the risk of cardiovascular disease. Overall, the treatment helps to restore normal physiological functions, which may work through mechanisms such as reducing oxidative stress, anti-inflammatory or improving organ repair. These improvements provide physiological support for the treatment effect, indicating that the treatment may have a beneficial effect on the body through multiple mechanisms.

[0132] Figure 5 Changes of biochemical indicators in AD mice

[0133] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0134] 3. Anti-inflammatory activity of Thuja sutchuenensis volatile oil

[0135] The levels of related inflammatory factors in the serum of mice treated with Thuja sutchuenensis volatile oil were detected. Figure 6 , Thuja volatile oil and positive drugs can significantly reduce the serum levels of IL-4, TNF-α, IFN-γ, and IgE (p≤0.05), and there is a decreasing trend for IL-10 and IL-1β, but it did not reach a significant level (p>0.05), and reached a level equivalent to that of the positive drug group, indicating that Thuja volatile oil has the effect of inhibiting inflammatory factors and may control the course of AD by reducing inflammatory responses.

[0136] Figure 6 Thuja volatile oil regulates the level of inflammatory factors

[0137] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0138] 4. Evaluation of the antioxidant capacity of Thuja sutchuenensis volatile oil

[0139] After Thuja volatile oil was used to treat AD mice, serum H2O2 concentration and total iron binding capacity were significantly increased. Figure 7 , Thuja volatile oil and positive drugs can reduce the level of AD mice after treatment, but 5% Thuja volatile oil treatment has the most significant effect (p ≤ 0.05), while 10% Thuja volatile oil has the same effect as positive drugs.

[0140] Figure 7 Serum antioxidant capacity level of Thuja sutchuenensis essential oil in the treatment of AD mice

[0141] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0142] 5. Alleviation of pathological state of AD mice

[0143] The mice in different treatment groups were subjected to histopathological sectioning and HE and toluidine blue staining. Figure 8 HE staining showed that the epidermal structure of the skin tissue was intact and clear. The epidermis of the mice in the model group was thickened, and the volume and number of prickle cells increased, which was significantly different from the control group and the 5% thuja treatment group (p≤0.05). A small amount or part of the collagen fibers in the dermis were degenerated and necrotic, the structure of the necrotic collagen fibers was blurred, and the nuclei were condensed and collapsed; the dermis was accompanied by varying degrees of inflammatory cell infiltration, mainly neutrophils with lobed nuclei or rod-shaped nuclei and lymphocytes with oval nuclei, while in the 5% thuja volatile oil treatment group, a small amount or part of the fibrous tissue in the dermis proliferated, the number of fibroblasts with oblong nuclei increased, and the arrangement of the proliferating fibroblasts was more disorderly. The subcutaneous fat layer and muscle layer structure were clearly visible, the fat cells and muscle fibers were neatly arranged, and the cell nuclear morphology was normal. Toluidine blue staining showed mast cells and degranulated mast cells. The model group had the most mast cells, which was significantly different from the control group (p≤0.01). After treatment with 5% Thuja sutchuenensis essential oil, the number of mast cells decreased (p>0.05), but did not return to a healthy level.

[0144] Figure 8 Pathological changes of AD mice

[0145] Note: CK, normal saline group, Model, OVA modeling; 5% YB, 5% Thuja sutchuenensis essential oil treatment after modeling, 10% YB, 10% Thuja sutchuenensis essential oil treatment after modeling. Compared with the model group, *p≤0.05, p≤0.01.

[0146] 6. Changes in skin microbial homeostasis in AD mice

[0147] The skin microorganisms of AD mice were sequenced and identified, and the changes in their microbial composition under different treatments were analyzed. Fig. 9 . The species composition showed differences between groups ( Fig. 9 A), Staphylococcus lentus was the dominant species in the Thuja volatile oil and blank groups, while Arthrobacter sp. was the dominant species in the model group; the overlap of species between the blank group and the Thuja volatile oil treatment group was high ( Fig. 9 B, C), the model group had more bacterial species and lower relative abundance, but the species composition diversity among the three groups was not significantly different ( Fig. 9 D), from the analysis of the ecological niche occupied by the bacterial species composition, the ecological niches among the three groups were significantly different ( Fig. 9 E). It can be seen that the skin microbial balance of AD mice was destroyed, and the bacterial species were rich, but the composition of skin microorganisms was quite different from that of healthy mice in the blank group. The mice treated with Thuja sutchuenensis volatile oil showed similar composition and abundance of skin microorganisms to those of healthy mice. Therefore, Thuja sutchuenensis volatile oil may play a therapeutic effect in treating AD by regulating the composition of skin microorganisms and maintaining the balance of skin microorganisms.

[0148] Fig. 9 Changes in skin microbial composition of AD mice; Note: A, species composition analysis, B, species composition Sankey diagram, C, species composition evolution tree analysis, D, α diversity analysis, E, niche analysis. CK, saline group, Model, OVA modeling; YB, treated with 5% Thuja volatile oil after modeling. *p≤0.05, p≤0.01.

[0149] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for constructing Thuja sutchuenensis volatile oil in the preparation of a product for treating skin diseases based on an animal model, characterized in that: The following steps are involved: (1) Healthy mice were selected as experimental animals and evenly divided into experimental group, control group and blank group according to their weight; (2) The allergic dermatitis model was established by repeatedly applying ovalbumin (OVA) solution to the skin, including the first application of OVA solution combined with adjuvant, followed by another application one week later, and continuous stimulation for 4 weeks; (3) During the induction process, observe the behavioral reactions of mice, such as skin redness, swelling, and scratching, and record the number of scratching times and the degree of skin damage; (4) After the model was constructed, the therapeutic product prepared from Thuja sutchuenensis volatile oil was applied to the damaged skin of mice in the experimental group to observe the therapeutic effect.

2. The construction method according to claim 1, characterized in that: The induction steps of the allergic dermatitis model include the following processes: (1) During the first application, the OVA solution (concentration of 10%) and complete Freund's adjuvant (CFA) were mixed and evenly applied to the shaved area on the back of the mouse; (2) After a one-week interval, the mouse skin was repeatedly smeared with 10% OVA solution every 3 days until the 28th day of the experiment; (3) Confirm whether the skin inflammation model has been successfully established in mice through visual observation and scratching behavior recording.

3. The construction method according to any one of claims 1 or 2, characterized in that: After the model is constructed, the success of the model is verified by the following methods: (1) Serum samples were used to detect the concentrations of inflammatory factors related to allergic dermatitis, including IgE, IL-4, IL-10, IL-1β, TNF-α, IFN-γ, etc.; (2) Observe mouse skin tissue sections by HE staining to confirm the degree of inflammatory cell infiltration and changes in epidermal thickness; (3) The oxidative stress level of mouse skin was measured and the H2O2 concentration and total iron binding capacity in serum were detected.

4. The construction method according to claim 1, characterized in that: The therapeutic effect of Thuja sutchuenensis volatile oil was analyzed by the following methods: (1) Record the changes in the number of scratching times of mice in the experimental group after treatment with Thuja sutchuenensis volatile oil, and compare the changes before and after treatment; (2) Analyze the differences in the microbial composition of mouse skin before and after treatment, and determine the types and diversity of microorganisms using 16S rRNA gene sequencing; (3) Immunohistochemical staining was used to observe the degree of skin tissue repair after treatment, including the reduction of inflammatory cell infiltration and the recovery of epidermal structure.

5. A use of Thuja sutchuenensis volatile oil in the preparation of a drug for treating skin diseases, characterized in that: The thuja volatile oil is used to prepare a medicine for treating skin allergic inflammation, and the medicine can achieve a therapeutic effect by inhibiting the release of inflammatory factors, regulating the composition of skin microorganisms and promoting skin tissue repair.

6. The use of Thuja sutchuenensis volatile oil as claimed in claim 5 in preparing a drug for treating skin diseases, characterized in that: The medicine is an external preparation, including ointment, cream, gel or spray form, in which the mass concentration of thuja volatile oil is 0.1% to 5%, and is mixed with a skin-friendly matrix.

7. The use of Thuja sutchuenensis volatile oil as claimed in claim 5 in preparing a drug for treating skin diseases, characterized in that: The drug is used to treat allergic dermatitis, eczema and skin infections by reducing the level of skin inflammatory factors, enhancing antioxidant capacity, and improving the balance of skin microbiota.

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