Use of a gaozuo oil tea extract and a medicament for treating atopic dermatitis

Abstract

The application discloses a use of Camellia koushattensis extract and a medicine for treating atopic dermatitis. The use of the Camellia koushattensis extract is used for preparing the medicine for treating atopic dermatitis; the Camellia koushattensis extract is Camellia koushattensis pressed tea oil, and the Camellia koushattensis pressed tea oil contains a characteristic active ingredient (1R, 3R, 9S)-3, 11, 11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one; the active ingredient is a caryophyllane-type sesquiterpene ketone, and the relative abundance of the active ingredient is significantly increased compared with ordinary Camellia oleifera seed oil. Based on a MC903-induced atopic dermatitis mouse model, the immunoregulatory effect of the Camellia koushattensis extract is systematically evaluated, experimental evidence is provided for clinical application, and it is proved that the Camellia koushattensis extract has significant anti-inflammatory activity and immunoregulatory potential, and is used as a natural skin protective agent and an auxiliary treatment. The medicine containing the Camellia koushattensis extract has obvious effects on treating atopic dermatitis of mice, and is beneficial to further application in clinical treatment.

Description

Technical Field

[0001] This invention relates to the use of Camellia oleifera extract from Gaozhou and a drug for treating atopic dermatitis, belonging to the field of pharmaceutical technology. Background Technology

[0002] Atopic dermatitis ( Atopic Dermatitis Alzheimer's disease (AD) is a chronic inflammatory skin disease with a high global prevalence and significant social and economic burden. Statistics show that AD affects approximately 15–20% of children and 2–10% of adults, and its incidence is continuously rising. It has been listed by the World Health Organization as one of the world's key non-communicable chronic diseases of concern. AD is characterized by typical relapse-remission cycles, long-term persistence, and a tendency to be difficult to treat. Patients often experience a decline in quality of life and increased psychological burden due to persistent itching, skin fissures, and sleep disturbances. In severe cases, long-term dependence on corticosteroids or immunosuppressive drugs can occur, leading to side effects and adherence problems. Therefore, there is an urgent need to develop natural, mild, safe, and long-term adjunctive or alternative treatment strategies.

[0003] In terms of pathological mechanisms, immune dysfunction is a core characteristic of Alzheimer's disease (AD). Patients and animal models exhibit a significant type 2 immune response (…). Type-2 inflammation Overactivation of Th2 cells, primarily driven by the release of inflammatory cytokines such as IL-4, IL-13, and IL-31, along with the involvement of multiple inflammatory mediators including IFN-γ and IL-17, ultimately leads to skin barrier damage, stratum corneum moisture loss, nerve ending sensitization, and the induction of severe itching. Therefore, immunomodulators and skin barrier repair agents have become hot research topics in recent years. The mouse MC903-induced model, due to its high reproducibility of Th2 immune characteristics and pathological manifestations, is considered a standard preclinical experimental system for studying AD mechanisms and intervention strategies.

[0004] This study systematically evaluated the immunomodulatory effects of Camellia oleifera oil in a mouse model of MC903-induced atopic dermatitis, providing experimental evidence for its clinical application. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a drug containing Camellia oleifera extract for the treatment of atopic dermatitis and the technical solution for its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: One use of Camellia oleifera extract from Gaozhou for the preparation of a drug for treating atopic dermatitis; The Gaozhou camellia oil extract is Gaozhou camellia pressed oil, which contains a characteristic active ingredient: (1R,3R,9S)-3,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one. This active ingredient is a caryophyllene-type sesquiterpene ketone, and its relative abundance is significantly increased compared to ordinary camellia seed oil.

[0007] The above-mentioned drugs for treating atopic dermatitis also have the following pharmacological effects: 1) improving skin thickening caused by dermatitis, reducing skin inflammatory infiltration, and repairing the damaged structure of the stratum corneum; 2) regulating the skin immune response and maintaining skin immune homeostasis; 3) inhibiting local inflammatory reactions of the skin.

[0008] The above-mentioned inhibition of local skin inflammation includes inhibiting the abnormal expression of inflammatory cytokines IL-4 and Th1-type cytokines IFN-γ, and stabilizing the expression levels of skin inflammatory factors.

[0009] The above-mentioned inhibition of local skin inflammation includes targeted regulation of Th2 immune cell inflammatory pathways and inhibition of Th2-type inflammation-mediated skin inflammatory damage.

[0010] A drug for treating atopic dermatitis, wherein the active ingredient of the drug is camellia oil pressed from Gaozhou, and the camellia oil pressed from Gaozhou contains a characteristic anti-inflammatory active ingredient: (1R,3R,9S)-3,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one.

[0011] To ensure efficacy, the Gaozhou camellia oil mentioned above is produced from the fruit of 8-10 year old Gaozhou camellia trees from Gaozhou City, Maoming City, Guangdong Province, through drying, crushing, steaming, pressing, and settling. Compared with ordinary camellia seed oil of the same age and from the same production area, the relative abundance of (1R,3R,9S)-3,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one is increased by more than 1000 times.

[0012] The above 1000-fold or higher values ​​are relative abundance ratios obtained by mass spectrometry detection through broad-targeted metabolomics, used to characterize the component differences between the two types of camellia seed oil, reflecting the specific enrichment characteristics of this characteristic active ingredient in Gaozhou camellia seed oil.

[0013] Unless otherwise specified in this application, and not explicitly defined or agreed upon in the prior art, all percentages in this application refer to mass percentages.

[0014] Any techniques not mentioned in this invention are based on existing technologies.

[0015] The present invention has the following beneficial effects: (1) Based on an MC903-induced atopic dermatitis mouse model, this application systematically evaluates the immunomodulatory effects of Camellia oleifera extract from Gaozhou, providing experimental evidence for its clinical application. It demonstrates that Camellia oleifera extract from Gaozhou possesses significant anti-inflammatory activity and immunomodulatory potential, as well as its characteristics as a natural skin protectant and adjuvant therapy. Drugs containing Camellia oleifera extract from Gaozhou have a significant effect on treating atopic dermatitis in mice, which is beneficial for its further application in clinical treatment.

[0016] (2) This application demonstrates that Gaozhou tea oil has a targeted effect on the treatment of atopic dermatitis in mice, which is of certain significance for the preparation of drugs with fewer side effects. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of an animal disease model.

[0018] Figure 2 Phenotypic diagram of atopic dermatitis in the ear of an animal.

[0019] Figure 3 A diagram illustrating the clinical characteristics of how tea oil intervention in Gaozhou significantly improves atopic dermatitis.

[0020] Figure 4 For the histopathological evaluation of mouse ear tissue.

[0021] Figure 5 The number of spleen cells in mice.

[0022] Figure 6 This diagram shows the expression of key immune cell subsets and inflammatory factors in the spleen. Detailed Implementation

[0023] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0024] In this embodiment, the Gaozhou camellia oil was provided by Zhongkai University of Agriculture and Engineering. The preparation method is as follows: Camellia oleifera fruits from 9-year-old Gaozhou camellia trees (Gaozhou City, Maoming City, Guangdong Province) were selected, and after natural drying to a moisture content of 8%~12%, the seeds were dehulled to obtain camellia seeds (with a particle size of about 2~5mm and no intact camellia seed kernels); the camellia seeds were crushed and steamed at 100℃ for 20 minutes; after steaming, the seeds were pressed into tea cakes and pressed to extract oil using a horizontal wedge press; the crude oil obtained by pressing was allowed to settle at room temperature for 24 hours, and the clear oil on the top layer was taken to obtain Gaozhou camellia oil (Gaozhou camellia seed oil).

[0025] Except for the fact that ordinary tea oil uses the fruit of 9-year-old ordinary tea trees (in the hilly area of ​​western Guangdong Province) as raw material, the rest of the preparation process is the same as that of Gaozhou tea oil.

[0026] Example 1

[0027] Atopic dermatitis mouse modeling and pathological examination

[0028] 1. Materials and Methods

[0029] 1.1 Laboratory Animals

[0030] The experimental animals used in this study were healthy female BALB / c mice, provided by the Laboratory Animal Center of Shanghai Jiao Tong University, aged 7–8 weeks. A total of 30 mice were randomly divided into 3 groups of 12 mice each: a healthy group (Blank), a control group (treated with ordinary tea oil), and a treated group (treated with Gaozhou tea oil). All animals were housed in an SPF (Specific Pathogen Free) environment at the Laboratory Animal Center of Shanghai Jiao Tong University, with the temperature maintained at 22±2°C, relative humidity at 50%–60%, a 12-hour light / dark cycle, and free access to standard laboratory mouse feed and water.

[0031] All animal handling and experimental procedures strictly adhered to laboratory animal ethics and welfare guidelines, were approved by the International Association of Laboratory Animals (IACUC) of Shanghai Jiao Tong University, and were conducted in accordance with the national "Regulations on the Management of Laboratory Animals" and the "Guide for the Care and Use of Laboratory Animals." To minimize animal suffering, humane measures were implemented during experiments, including environmental decompression, handling mitigation, and terminal disposal. The experimental design followed the "3R principle" (Replacement, Reduction, Refinement), with reasonable planning of animal numbers and experimental procedures to ensure scientific rigor and ethical compliance.

[0032] 1.2 Experimental Materials and Main Reagents

[0033] (1) Modeling drugs and sample processing materials

[0034] Calcipotriol (MC903, MedChemExpress, MCE; Cat. No. HY-10001) was used to induce a mouse model of atopic dermatitis; anhydrous ethanol (analytical grade, Sinopharm Group) was used as the carrier solvent for MC903; Gaozhou tea oil and ordinary tea oil were used for topical intervention; 10% neutral formalin buffer (Solarbio) was used for skin tissue fixation; 75% ethanol (prepared with DEPC water) was used for sterilization during the experiment.

[0035] (2) General biological laboratory consumables

[0036] RNase-free microtubes (Axygen); phosphate-buffered saline (PBS, pH 7.4; Gibco); ACK erythrocyte lysis buffer (Ammonium-Chloride-Potassium lysis buffer; ThermoFisher); Cytofix™ fixative (BD Biosciences); 70μm cell strainer (Corning) for preparing single-cell suspensions from tissues; 100μL micropipette and sterile pipette tips (Eppendorf); sterile cotton swabs, ice box, 3mL syringe (disposable, medical grade).

[0037] (3) Antibodies and reagents related to flow cytometry

[0038] CD45-FITC monoclonal antibody (BioLegend); CD4-BV605 monoclonal antibody (BD Horizon™); Live / Dead™ BV421 live cell dye (BD Biosciences); IFN-γ-APC monoclonal antibody (BioLegend); IL-4-PE / Cy7 monoclonal antibody (BD Biosciences). (4) Experimental instruments and equipment Flow cytometer (BD FACSCanto™ II); micrometer (for ear thickness measurement); clean bench, CO2 incubator, electronic balance, refrigerator (4℃ / -20℃) and other basic experimental equipment.

[0039] 1.3 Establishment of an MC903-induced atopic dermatitis model

[0040] The design of animal disease model groups and treatment groups, such as Figure 1 As shown. MC903 was dissolved in anhydrous ethanol to prepare a working solution with a final concentration of 2 nmol / 20 μL, which was then aliquoted into 50 μL portions and stored at -20°C protected from light (for no more than one month). On Day 0, the initial ear thickness of both groups of mice (control group and treatment group) was measured and their body weight was measured. Subsequently, 20 μL of MC903 working solution was applied to the skin of the lateral (back of the ear) of the left ear of both groups of mice daily. The mice were then fixed and allowed to air dry naturally for 20 seconds. This process was repeated for 9 consecutive days to establish an atopic dermatitis model. On Day 9, the left ear of the mice showed obvious dermatitis symptoms; the healthy mice received no treatment.

[0041] Subsequently, every other day, 20 μL of MC903 working solution was applied to the skin on the outer side (behind the ear) of the left ear of the control group mice. Simultaneously, 20 μL of ordinary tea oil was applied to the left ear daily. After application, the mice were fixed and allowed to air dry for 20 seconds until the experiment ended on Day 18. For the treatment group mice, every other day, 20 μL of MC903 working solution was applied to the skin on the outer side (behind the ear) of the left ear. Simultaneously, 20 μL of Gaozhou tea oil was applied to the left ear daily until the experiment ended on Day 18. An equal volume of anhydrous ethanol was applied to the right ear.

[0042] 2. Detection Indicators and Experimental Methods

[0043] (1) Assessment of clinical signs

[0044] During the experiment, mouse body weight, ear swelling (measured with a micrometer), and the severity of dermatitis and itching were recorded. Dermatitis severity was assessed using a scoring system: erythema (0 = no erythema; 1 = mild: localized pale red patches on the auricle with indistinct borders; 2 = moderate: markedly red auricle, with erythema covering most of the auricle; 3 = severe: deep red or even purplish-red auricle with widespread congestion), edema / thickness (0 = no edema, skin thickness no different from normal mice; 1 = mild: slight swelling, slightly thickened skin, minimal changes in contour; 2 = moderate: markedly swollen and thickened auricle with increased wrinkles; 3 = severe: significantly thickened and bulging auricle with deformed shape), and epidermal shedding (0 = no desquamation, 1 = desquamation present). Long-distance and macro photography was taken of both ears of the mice at all assessment points.

[0045] (2) Histopathological examination (HE staining)

[0046] Mice were sacrificed at the end of Day 18 of the experiment, and the sacrifice was carried out in accordance with animal ethics requirements and humane terminal disposal. Immediately afterwards, auricular tissue was taken from both ears. The tissue was cut with sterile scissors and a circular ear tissue sample of the same diameter was obtained using an ear tissue punch to ensure the consistency of tissue section area between different animals and different groups and to reduce the bias caused by the difference in cutting. The obtained ear tissues were quickly fixed in pre-cooled 10% neutral formalin buffer for 48 hours (room temperature), then embedded in paraffin as usual, and sectioned continuously in a direction perpendicular to the skin surface (thickness 4–5 μm). After dewaxing and hydration, the sections were stained with standard hematoxylin-eosin (H&E) staining procedure, and then mounted after dehydration and clearing. After staining, the sections were observed under an optical microscope and pathologically evaluated. (3) Immune cell subset analysis (flow cytometry)

[0047] Immediately after euthanizing the mice at the end of Day 18, spleen tissue was harvested and placed in pre-chilled RNase-free EP tubes. The spleen was gently homogenized in a clean bench using a 70 μm cell sieve, and the cell suspension was collected by slow washing with pre-chilled PBS. The supernatant was then removed by centrifugation at 500×g for 5 min, and 2 mL of ACK erythrocyte lysis buffer was added and incubated at room temperature for 3 min to lyse the erythrocytes. PBS was then added to terminate the lysis reaction, and the cells were centrifuged again to remove residual liquid. The resulting cell pellet was resuspended in 0.5 mL of PBS, filtered, and counted using a cell counting chamber (100-fold dilution). 1×10⁻⁶ cells were collected. 7 The cells were used for subsequent staining analysis.

[0048] Surface labeling staining was performed using an antibody-mixed staining solution prepared with PBS, comprising CD45-FITC (1:150 dilution), CD4-BV605 (1:150 dilution), and Live / Dead™ BV421 survival dye (1:10000 dilution). 50 μL of the antibody mixture was added to each sample, and the cells were incubated at 4°C in the dark for 20 min. After incubation, cells were washed with PBS and centrifuged to remove unbound antibodies. 150 μL of Cytofix™ fixative was then added, and the cells were incubated at room temperature in the dark for 20 min to fix them. After fixation, the cells were washed with Cytofix Wash Buffer and resuspended.

[0049] Intracellular cytokine staining was then performed: IL-4-PE / Cy7 (1:150 dilution) and IFN-γ-APC (1:150 dilution) antibodies were added, and the cells were incubated at room temperature in the dark for 20 min. After final washing, the cells were resuspended in 200 μL PBS and immediately analyzed by flow cytometry (BD FACSCanto™ II). Subsequent data analysis was performed using FlowJo software.

[0050] Example 2

[0051] Intervention with tea oil from Gaozhou significantly improved the phenotype of atopic dermatitis in the ears of mice.

[0052] like Figure 2 As shown, starting from the 9th day of continuous MC903 application (Day 9), both the control and treatment groups of mice exhibited typical clinical manifestations of atopic dermatitis in their left ears, including significant thickening of the auricle, erythema, edema, dryness and desquamation, and focal epidermal peeling, indicating successful model establishment. With continued stimulation, although ordinary tea oil was used for intervention later, the inflammatory symptoms in the control group showed a continuous worsening trend, reaching the peak of the disease course on Day 18, with both ear thickness and skin lesion severity at their most severe state.

[0053] In contrast, although the treated mice also showed obvious skin lesions in the early stages (Days 9–12), the disease progression curve exhibited a dynamic pattern of initial increase followed by decrease. Specifically, the degree of inflammation in the treated group was basically consistent with that in the control group before Day 12, reaching its peak. However, under the intervention of Camellia oleifera oil, the symptoms began to significantly improve from Day 14, manifested as reduced ear thickness, gradual fading of erythema, and a significant reduction in desquamation and peeling. By Day 18, the appearance of the ear skin in the treated group had basically recovered to near-normal levels, with only a small amount of mild erythema remaining.

[0054] In summary, Gaozhou camellia oil can significantly delay and reverse the progression of MC903-induced atopic dermatitis. Its improvement trend and time points are clearly identifiable, providing phenotypic evidence for subsequent histological and immunological index analysis. It also suggests that it has potential anti-inflammatory and skin barrier repair effects, and its effects are significantly better than those of ordinary camellia oil.

[0055] Example 3

[0056] Intervention with Gaozhou camellia oil significantly improved the clinical characteristics of atopic dermatitis.

[0057] like Figure 3 As shown, this study comprehensively evaluates the therapeutic effect of Gaozhou camellia oil from two dimensions: overall physiological state and local skin damage.

[0058] First, to rule out potential systemic side effects from the intervention oil itself, this study monitored the dynamic changes in mouse body weight during the experiment, such as... Figure 3 A. The results showed that, regardless of whether it was the control group or the treatment group, the body weight of mice showed a slight increasing trend over time, and the differences at each time point were not statistically significant. P >0.05), indicating that external application of Gaozhou tea oil will not cause significant metabolic burden or systemic toxicity, and has good safety.

[0059] In terms of clinical skin lesion phenotype, Gaozhou tea oil showed a significant alleviating effect. Ear thickness, as a quantitative indicator reflecting inflammatory edema and dermal hyperplasia, provides the most direct visual indication of dynamic changes. Figure 3 B). In the control group, ear thickness increased rapidly from Day 9 and continued to accumulate until Day 18. In the treatment group, although the increase in ear thickness was similar in the early stage of modeling (Days 9–12), a decreasing inflection point appeared from Day 14, and the difference from the control group widened significantly. By Day 18, ear thickness was close to normal levels. P The result was <0.01, indicating that Gaozhou tea oil can effectively inhibit MC903-induced epidermal thickening and inflammatory edema.

[0060] In addition, the redness and swelling score ( Figure 3 C) and scaling (scaling) score ( Figure 3D) Further reflects the degree of local skin inflammation and barrier damage. From Day 9 onwards, the control group showed a typical worsening trend of inflammation, with bright erythema and extensive desquamation on the ears. In contrast, with the continued intervention, the redness and swelling in the treatment group gradually decreased and were significantly relieved, and the scaling and peeling also improved significantly, indicating that Gaozhou tea oil has a repairing and protective effect on epidermal dryness and barrier damage.

[0061] Based on the combined quantitative indicators of skin lesions and body weight data, Gaozhou camellia oil has good safety and significant local anti-inflammatory and analgesic effects. It can significantly improve the inflammatory phenotype of ear skin in AD model mice and promote the gradual recovery of damaged skin tissue, with effects significantly better than ordinary camellia oil.

[0062] Example 4

[0063] Intervention with tea oil from Gaozhou significantly improved the clinical characteristics of atopic dermatitis.

[0064] like Figure 4 As shown, morphological observation of ear tissue by H&E staining further verified the histological ameliorative effect of Gaozhou tea oil on MC903-induced atopic dermatitis.

[0065] In the control group (right ear), the ear skin structure was intact, the epidermis was of uniform and normal thickness, the stratum corneum was continuous, no obvious inflammatory cell infiltration was observed in the dermis, and the skin appendages remained intact, indicating that the mice had healthy skin tissue without inflammatory stimulation. Figure 4 Left).

[0066] The ear tissue of control mice (treated with ordinary tea oil) exhibited typical pathological features of atopic dermatitis. The epidermis was significantly thickened, with hyperkeratosis, blurred epidermal-dermal interface, and abundant infiltration of inflammatory cells, including lymphocytes and eosinophils, in both the dermis and epidermis, accompanied by local edema and vasodilation, indicating significant local tissue damage. Figure 4 (In the middle). These pathological changes are highly consistent with clinical symptoms (erythema, swelling, desquamation), indicating that the model was successfully established and the inflammatory response was active.

[0067] After treatment with Gaozhou camellia oil, the experimental group of mice showed significant improvement in ear histopathology. The epidermis was significantly thinned, the keratinized layer became more uniform, the number of inflammatory cells infiltrating the dermis and epidermis decreased, local edema was relieved, and the tissue structure recovered well. Figure 4 (Right) This suggests that Gaozhou tea oil can effectively alleviate MC903-induced skin inflammatory damage.

[0068] Overall, the histological results were highly consistent with the clinical phenotypic improvement, further supporting the local anti-inflammatory effects and barrier repair potential of Camellia oleifera oil on the skin of AD model mice. This finding suggests that its mechanism of action may involve inhibiting local inflammatory cell infiltration, reducing epidermal thickening and abnormal keratinization, thereby alleviating pathological damage and improving skin barrier function.

[0069] Example 5

[0070] Number of spleen cells in mice in different treatment groups

[0071] like Figure 5 As shown, on Day 18, mice were sacrificed and spleen tissue was obtained. After mechanical grinding, filtration through a 70 μm membrane, and removal of red blood cells using ACK lysis buffer, a single-cell suspension was obtained, and cell counting was performed to assess whether the intervention with Gaozhou tea oil induced a systemic immune response or caused changes in the number of peripheral immune cells. The results showed that the number of spleen cells remained at similar levels in both the control and treatment groups, with no statistically significant difference between the groups. P The result was >0.05, indicating that under the experimental conditions, continuous external application of Gaozhou tea oil for 9 days did not cause significant changes in splenic lymphocyte proliferation or apoptosis.

[0072] From an immunological perspective, the spleen is an important organ for the reserve and stress regulation of peripheral immune cells. Changes in its total cell count are often used to determine whether treatment induces systemic immune hyperactivity or immunosuppression. In the MC903-induced atopic dermatitis model, the main inflammatory response was limited to the local skin, without significant expansion or reduction of spleen cells, suggesting that the model did not cause significant systemic immune imbalance during the experimental period.

[0073] More importantly, the intervention with Gaozhou camellia oil did not cause abnormal changes in peripheral immune cells, reflecting that its anti-inflammatory effect is more likely concentrated in local skin regulation rather than through systemic immunosuppression. In other words, Gaozhou camellia oil has the characteristic of skin-targeted local regulation, exerting anti-inflammatory effects and improving skin lesions without interfering with the body's overall immune homeostasis, further validating its safety profile.

[0074] In summary, the stable number of spleen cells not only eliminates the potential immunosuppressive side effects that may be caused by Gaozhou tea oil, but also supports its therapeutic advantages of local anti-inflammatory and systemic safety, providing important background evidence for subsequent analysis of immune cell subsets and mechanism research.

[0075] Example 6

[0076] Splenic immune cell subset analysis

[0077] like Figure 6As shown, this study analyzed the expression of key immune cell subsets and inflammatory factors in the spleen using flow cytometry to assess the potential impact of Gaozhou camellia oil on the immune system in vivo. The results showed no significant difference in the expression levels of Th2-related inflammatory cytokines IL-4 and Th1-related cytokines IFN-γ in the spleen of mice between the control group and the Gaozhou camellia oil treatment group. P >0.05), indicating that under experimental intervention conditions, MC903 modeling and tea oil treatment did not induce significant systemic immune activation or inhibition responses.

[0078] From an immunological perspective, the spleen is an important reservoir of peripheral immune cells, and abnormalities in its cytokine expression and subset ratios usually reflect changes in the systemic immune response. However, the data from this experiment show that while topical application of Camellia julibrissin oil significantly alleviated local skin inflammation and histopathological damage, it did not induce the expansion or inhibition of Th1 / Th2 immune cells in the spleen. This indicates that the anti-inflammatory effect of Camellia julibrissin oil is mainly limited to the local skin and does not interfere with the body's systemic immune homeostasis, demonstrating its localized, low-risk immunomodulatory characteristics.

[0079] Furthermore, this result is consistent with previous analyses of total spleen cell count ( Figure 5 This finding is consistent with the findings, further validating that Gaozhou camellia oil exerts its skin-protective effect by locally regulating the Th2 inflammatory pathway while maintaining the body's immune balance. This characteristic is of great significance for clinical application: it can improve the symptoms of atopic dermatitis while avoiding the risks of systemic immunosuppression or side effects, providing experimental evidence for Gaozhou camellia oil as a natural topical therapeutic agent.

[0080] This study established atopic dermatitis in BALB / c mice based on MC903 induction. Atopic Dermatitis The anti-inflammatory effect and immunomodulatory mechanism of Gaozhou camellia oil in atopic dermatitis were systematically explored using the AD model.

[0081] First, a mouse model of Alzheimer's disease (AD) with typical phenotypes such as skin erythema, scaling, thickening, and scratching behavior was established by continuous external stimulation of the auricle with MC903. The mouse was then treated with camellia oil from Gaozhou. The efficacy of camellia oil in improving skin symptoms was evaluated through clinical signs (ear thickness measurement, degree of skin redness and swelling, dryness and desquamation), body weight, and behavioral monitoring. Subsequently, from a histopathological perspective, this study further assessed the effects of camellia oil on skin structure repair and inflammatory damage relief by observing epidermal hyperplasia, dermal inflammatory cell infiltration, and stratum corneum integrity using hematoxylin and eosin (H&E) staining. At the immunological level, flow cytometry was used to detect changes in immune cell subsets in the spleen.

[0082] This study systematically explored the therapeutic effect of Gaozhou camellia oil in an MC903-induced BALB / c mouse model of atopic dermatitis (AD) by establishing a multi-dimensional evaluation system encompassing clinical phenotype, histopathology, and immune cell subset analysis. Experimental results showed that topical intervention with Gaozhou camellia oil significantly improved the skin inflammation manifestations in the model mice. Clinically, the treated group showed significant relief of ear erythema, swelling, desquamation, and edema compared to the control group, with significant decreases in ear thickness and skin lesion scores. Histologically, H&E staining revealed a significant reduction in epidermal thickness, decreased infiltration of inflammatory cells in the dermis and epidermis, and a tendency for restoration of the stratum corneum structure, effectively reversing the degree of pathological damage.

[0083] Further immunological analysis showed that intervention with Gaozhou camellia oil did not cause significant changes in the total number of spleen immune cells or Th1 / Th2-related inflammatory factors (IFN-γ, IL-4), suggesting that its anti-inflammatory effect is mainly limited to local skin tissue, improving local inflammation without interfering with systemic immune homeostasis. This characteristic reflects the advantages of Gaozhou camellia oil in localized targeted anti-inflammatory effects and high safety, providing experimental evidence for its application in atopic dermatitis and other chronic inflammatory skin diseases.

[0084] In summary, this study is the first to systematically demonstrate that Gaozhou camellia oil has significant anti-inflammatory and tissue-protective effects on an atopic dermatitis model. Its mechanisms may include inhibiting local inflammatory cell infiltration, reducing epidermal thickening, and improving keratinization abnormalities. The findings not only provide a scientific basis for the clinical functionalization development of Gaozhou camellia oil but also offer new ideas and potential strategies for the application of natural plant oils in the prevention and treatment of chronic inflammatory skin diseases.

[0085] Example 7

[0086] To explore the material basis for the effect of Gaozhou camellia oil in improving dermatitis, this embodiment compares the differences in metabolite composition between Gaozhou camellia oil and ordinary camellia oil.

[0087] Metabolomics analysis using broad-targeted metabolomics revealed a characteristic compound significantly upregulated in Gaozhou camellia seed oil: (1R,3R,9S)-3,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one. This compound belongs to the caryophyllene-type sesquiterpene ketones; its normalized abundance in Gaozhou camellia is as high as approximately 5.03 × 10⁻⁶. 6In contrast, the level of sesquiterpenoids in ordinary camellia oil is only about 5.4 × 10³, representing an average upregulation of 926.5 times (Log2FC = 9.86, P = 0.0049). This makes it one of the most differentially expressed terpenoid components among all 1173 differentially expressed metabolites, and it ranks first in Log2FC among the 65 sesquiterpene metabolites. More importantly, this difference is not an isolated event: the sesquiterpene group as a whole is systematically activated—56 of the 65 sesquiterpenes are significantly upregulated in Gaozhou camellia oil (47 of which have P < 0.05), including caryophyllene skeleton derivatives such as 6-acetoxycaryophyllidiene, as well as sesquiterpene ketones with phytoseptic activity such as naringinone and Africanone. This suggests that Gaozhou camellia oil has evolved a "chemical defense" metabolic characteristic not found in ordinary camellia oil. Combining the known activities of these components, such as disrupting fungal cell membranes and activating CB2 receptors to exert peripheral anti-inflammatory and analgesic effects, this "component difference" directly endows Gaozhou camellia oil with a differentiated development value that far exceeds that of ordinary camellia oil in terms of disease resistance, anti-inflammatory and analgesic effects, antibacterial properties, and high-end daily chemical applications, forming a complete differentiation chain from metabolic characteristics to function to industrial value.

[0088] Broadly targeted metabolome differential metabolite expression

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] In the table, B3-1 and B3-2 represent the peak areas of Gaozhou camellia oil; B1-1 and B1-2 represent the peak areas of ordinary camellia oil; each group consists of two parallel experimental groups. P-valuation is the p-value, indicating statistical significance; the smaller the p-value, the more reliable the difference between groups. FDR is the corrected p-value. Fold_Chan is the fold change in FC (mean of B3 / mean of B1); FC > 1 indicates upregulation, FC < 1 indicates downregulation. Log2F is Log2(FC), the fold change in log2 transformation; Log2FC > 0 indicates upregulation, Log2FC < 0 indicates downregulation. Type of difference: up indicates significant upregulation; down indicates significant downregulation.

Claims

1. The use of a Gaozhou camellia oil extract, characterized in that: Used in the preparation of drugs for the treatment of atopic dermatitis; The Gaozhou camellia oil extract is Gaozhou camellia pressed oil, which contains a characteristic active ingredient: (1R,3R,9S)-3,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one. This active ingredient is a caryophyllene-type sesquiterpene ketone, and its relative abundance is significantly increased compared to ordinary camellia seed oil.

2. The use of the Camellia oleifera extract according to claim 1, characterized in that: The drug has the following pharmacological effects: 1) improves skin thickening caused by dermatitis, reduces skin inflammatory infiltration, and repairs the damaged structure of the stratum corneum; 2) regulates the skin immune response and maintains skin immune homeostasis; 3) inhibits local inflammatory response of the skin.

3. The medicinal use of the Camellia oleifera extract according to claim 2, characterized in that: The inhibition of local skin inflammation includes suppressing the abnormal expression of inflammatory cytokines IL-4 and Th1-type cytokines IFN-γ, and stabilizing the expression levels of skin inflammatory factors.

4. The medicinal use of the Camellia oleifera extract according to claim 2, characterized in that: The inhibition of local skin inflammation includes targeted regulation of Th2 immune cell inflammatory pathways and inhibition of Th2-mediated skin inflammatory damage.

5. A drug for treating atopic dermatitis, characterized in that: The active ingredient of the drug is Gaozhou camellia oil, which contains a characteristic anti-inflammatory active ingredient: (1R,3R,9S)-3,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one.

6. The medicament for treating atopic dermatitis according to claim 5, characterized in that: The Gaozhou camellia oil is produced from the fruit of 8-10 year old Gaozhou camellia trees from Gaozhou City, Maoming City, Guangdong Province, through drying, crushing, steaming, pressing, and settling. Compared with ordinary camellia seed oil of the same age and from the same production area, the relative abundance of (1R,3R,9S)-3,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-en-2-one is increased by more than 1000 times.

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