Application of composition in preparation of medicine for preventing inflammatory aging and related diseases thereof

Abstract

The invention relates to the technical field of biology, in particular to application of a composition in preparation of drugs for preventing inflammatory aging and related diseases thereof. The composition provided by the invention is used for preventing atopic dermatitis (AD) recurrence, senile dermatitis and other BCAA metabolic disorder related skin diseases by selectively enhancing BCAT1 mediated branched chain amino acid (BCAA) degradation and reversing skin inflammatory aging. Clinical data show that after the composition provided by the invention is used for treatment, the recurrence rate of AD is reduced to 16.3% from 35.7%. The invention provides a non-hormone replacement therapy, which makes up the defects of the existing medicines, and the composition can be combined with the existing therapy during treatment, has high safety and no serious adverse reaction, and is suitable for long-term maintenance treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the application of compositions in the preparation of preventives against inflammatory aging and related diseases. Background Technology

[0002] Inflammatory aging is a core pathological mechanism in many chronic skin diseases, such as atopic dermatitis, characterized by elevated expression of cellular senescence markers (such as P21) and the release of senescence-associated secretory phenotype (SASP) factors. Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease affecting approximately 200 million people worldwide (Tian et al., 2023). Its clinical features include intense itching and eczematous lesions, and its pathogenesis involves a complex interaction of epidermal barrier dysfunction, immune dysregulation, and environmental factors (Guttman-Yassky et al., 2025). A typical feature of AD is a type 2 inflammatory response mediated by cytokines IL-4, IL-5, and IL-13, leading to abnormal keratinocyte function, itching, and impaired barrier integrity (Facheris et al., 2023). Current treatments (such as topical corticosteroids and calcineurin inhibitors) can effectively control acute flare-ups, but often fail to prevent relapse and have potential adverse reactions (Stefanovic and Irvine, 2024). Therefore, there is an urgent need to develop safe and effective therapies to prevent AD relapse, target type 2 inflammatory drivers, and improve long-term disease control. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide the application of the composition in the preparation of a preventive for inflammatory aging and related diseases. The composition provided by the present invention reverses inflammatory aging of the skin by selectively enhancing the degradation of branched-chain amino acids (BCAAs) mediated by BCAT1, and is used to prevent the recurrence of atopic dermatitis (AD), senile dermatitis and other skin diseases related to BCAA metabolic disorders.

[0004] This invention provides the use of the composition in the preparation of products for the prevention and / or treatment of diseases caused by the overexpression of branched-chain amino acids BCAA, wherein:

[0005] The composition comprises the following components in parts by weight:

[0006] 20-40 parts of hemp seed, 20-40 parts of angelica, 5-15 parts of notoginseng, 10-30 parts of chuanxiong, 20-40 parts of coix seed, and 20-40 parts of licorice.

[0007] In some embodiments, the composition comprises the following components in parts by weight:

[0008] 30 parts hemp seed, 30 parts angelica root, 10 parts Panax notoginseng, 15 parts Ligusticum chuanxiong, 30 parts coix seed, and 30 parts licorice root.

[0009] In some embodiments, the disease includes at least one of skin diseases, metabolic diseases, neurological diseases, and age-related diseases.

[0010] In some embodiments, the skin disease includes Th2 cell-mediated skin diseases.

[0011] In some embodiments, the disease is selected from at least one of atopic dermatitis, senile pruritus, photoaging, psoriasis, chronic eczema, type 2 diabetes, obesity, non-alcoholic fatty liver disease, hepatic encephalopathy, maple syrup diabetes, frailty syndrome, and cognitive decline.

[0012] In some embodiments, the composition upregulates the expression of branched-chain aminotransferase 1.

[0013] This invention addresses the issue that the roles of BCAA metabolic enzymes BCAT1 and BCAT2 in the skin are not clearly distinguished, and existing technologies lack specific activation strategies for BCAT1. The invention discovers that the composition selectively activates the BCAT1 enzyme, promotes BCAA degradation, reverses inflammatory aging, and further achieves precise regulation of skin metabolism and inflammatory aging.

[0014] In some embodiments, the composition inhibits the expression of the aging marker P21.

[0015] In some embodiments, the composition inhibits the expression of the aging-related secretory phenotype factor SASP.

[0016] In some embodiments, the product includes pharmaceuticals and / or beauty products.

[0017] In some embodiments, the product is any one of emulsion, oil, gel, cream, lotion, spray, sustained-release formulation, or controlled-release formulation.

[0018] Beneficial effects:

[0019] 1. This invention has discovered that the composition of the Six Flavors Moisturizing Oil can enhance BCAA catabolism and reduce BCAA accumulation and SASP factor release by selectively upregulating BCAT1 expression (rather than BCAT2).

[0020] 2. This invention has discovered that the composition of the Six Flavors Moisturizing Oil can simultaneously inhibit Th2 cell activation and inflammatory aging markers (P21), thereby achieving a synergistic effect of metabolism and immunity.

[0021] 3. This invention selectively activates the BCAT1 enzyme, promotes BCAA degradation, and reverses inflammatory aging. It is applicable to the prevention of AD recurrence, senile pruritus, photoaging, and other BCAA metabolic disorders. Clinical data show that after treatment with the composition provided by this invention, the recurrence rate of AD decreased from 35.7% to 16.3%.

[0022] 4. This invention provides a non-hormone replacement therapy that overcomes the shortcomings of existing drugs. The composition can be used in combination with existing therapies during treatment, has high safety, no serious adverse reactions, and is suitable for long-term maintenance therapy. Attached Figure Description

[0023] Figure 1 The characteristic GC-MS total ion chromatogram of LWRF oil (i.e., Liuwei Mofang oil) is shown.

[0024] Figure 2 The CONSORT flowchart illustrates the patient screening, randomization, allocation, follow-up, and analysis process.

[0025] Figure 3 This study demonstrates that LWRF oil prevents recurrence of skin lesions during the remission period of atopic dermatitis and improves clinical outcomes. Figure A compares the recurrence rates at weeks 4 and 8 between the LWRF oil treatment group (n=43) and the La-cer emulsion control group (n=42). Figure B shows the changes in Eczema Area and Severity Index (EASI) over time in both the LWRF oil and La-cer treatment groups. Figure C shows the changes in Investigator Global Assessment (IGA) scores over time. Figure D shows the changes in Dermatology Quality of Life Index (DLQI) scores over time. Figure E compares the baseline and week 4 serum IgE levels between the treatment and control groups. *p<0.05, **p<0.01, and ***p<0.001 vs. the control group;

[0026] Figure 4 This study investigated the targeted metabolomics analysis of skin swabs from healthy controls (HC, n=9), AD patients (AD, n=9), and AD patients treated with LWRF oil (LWRF, n=9) by modulating BCAA metabolism to reshape the skin metabolome in atopic dermatitis. Figure A shows the PCA score plot, indicating the separation of metabolic clusters among the three groups (PC1 explains 30.4% of the variance, PC2 explains 18.2% of the variance). Figures B and C show the relative abundance of metabolite categories. Figure D shows the pathway enrichment analysis, revealing significantly altered metabolic pathways in the AD group (highlighting the BCAA degradation pathway). Figure E is a heatmap of monomeric metabolite abundance: BCAA metabolite levels were elevated in AD skin and decreased after LWRF oil treatment. Figure F is a box plot showing the changes in valine, leucine, and isoleucine levels in the three groups. *p<0.05, **p<0.01 vs. the AD group.

[0027] Figure 5 The study showed that skin BCAA levels are associated with inflammatory aging in AD patients. Figure A shows a representative histopathological image of skin lesions in AD patients, showing epidermal hyperplasia, cavernous dermatitis, dermal inflammation, and collagen structure disorder. Figure B shows the immunohistochemical staining of P21 in skin lesions of AD patients (n=8) and healthy controls (n=6). Figure C shows the correlation between skin BCAA levels (valine, leucine, and isoleucine) and age in AD patients (n=9).

[0028] Figure 6 This study demonstrates that LWRF oil improves inflammatory aging in calcipotriol (MC903)-induced AD lesions by regulating BCAA metabolism. Figure A shows the quantitative analysis of BCAA in skin tissue based on the WST-8 colorimetric reaction; Figure B shows the levels of SASP factors IL-8 and IL-11; Figures C and D show the expression levels of P21 and BCAA metabolic enzymes (BCAT1 and BCAT2) in each group analyzed by immunohistochemistry and Western blot. *p<0.05,**p<0.01,***p<0.001,****p<0.0001 vs. the MC903 group;

[0029] Figure 7 This study illustrates how LWRF regulates Th2 cell responses induced by MC903 in atopic dermatitis. Figure A shows the gating strategy used in flow cytometry analysis; Figure B shows the percentage of CD45+ cells in live cells; Figure C shows the percentage of CD3+ cells in CD45+ cells; Figure D shows the percentage of CD4+ cells in CD3+ cells; and Figure E shows the percentage of GATA3+ cells in CD4+ cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. the MC903 group. Detailed Implementation

[0030] This invention provides the application of the composition in the preparation of a product for the prevention of inflammatory aging and related diseases. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0032] Example 1: Skin Metabolic Regulator

[0033] Composition of the regulator: This oil contains the following ingredients: hemp seed (30g), angelica (30g), notoginseng (10g), chuanxiong (15g), coix seed (30g), and licorice (30g). The six medicinal materials are pulverized into fine powder according to the proportions in Table 1, soaked in twice the volume of soybean oil for 7 days (stirring intermittently), filtered, and then packaged.

[0034] Table 1

[0035]

[0036]

[0037] Qualitative and quantitative analysis of compounds in LWRF oil was performed using gas chromatography-mass spectrometry (GC-MS). Within a 48-minute analysis period, 40 variables were detected based on retention time, mass-to-charge ratio (m / z), and peak intensity. Metabolite identification results are listed in Table 2, mainly including esters, alcohols, fatty acids, and other compounds. Other compounds showed weak signals or were not included in the database. The characteristic GC-MS total ion chromatogram of LWRF oil is shown below. Figure 1 GC-MS identification revealed key active ingredients such as Trilinolein (CAS 537-40-6) and Z-Butylidenephthalide (CAS 72917-31-8).

[0038] Table 2

[0039]

[0040]

[0041] Example 2 Skin Metabolomics Analysis

[0042] 1. Experimental Methods

[0043] This study was a single-center randomized controlled trial, approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine affiliated to Shanghai University of Traditional Chinese Medicine (approval number: 2024-099), and registered with the Chinese Clinical Trial Registry on May 24, 2024 (registration number: ChiCTR2400084762). Patient enrollment was from late June 2024 to January 2025.

[0044] Randomization sequences were generated by an independent statistician using SAS software (v9.4; SAS Institute Inc.). A stratified block randomization method was employed, maintaining a 1:1 allocation ratio with block sizes of 4–6. To ensure allocation concealment, a centralized, password-protected Interactive Web Response System (IWRS) was used. Researchers could only access treatment allocation information via IWRS after recruiting participants and entering and confirming baseline data. Due to inherent differences in appearance and texture between the two topical interventions (LWRF oil and La-cer body lotion), complete blinding of participants and treatment researchers was not feasible. Therefore, this study employed an assessor-blinded design, meaning that only outcome assessors were unaware of their group assignments.

[0045] Based on the preliminary results, the primary outcome of this study showed that the recurrence rate in the Liuwei Moisturizing Oil (LWRF) experimental group was approximately 20%, while the recurrence rate in the Yuze Skin Barrier Repairing Body Lotion control group was approximately 40%. With a significance level (α) of 0.05 and a power (β) of 0.1, the sample size estimation formula for comparing two rates under a completely randomized design was used:

[0046]

[0047] The calculated sample size for each group was n1 = n2 = 40. To account for subject non-compliance and dropout, the sample size was increased by 20%. Therefore, this study plans to recruit 96 subjects diagnosed with atopic dermatitis.

[0048] 1.1 Subjects

[0049] According to the Chinese Guidelines for the Diagnosis and Treatment of Atopic Dermatitis (2020 Edition), patients aged ≥18 years who were clinically diagnosed with moderate to severe AD and had an Investigator Global Assessment (IGA) score ≥3 were included.

[0050] 1.2 Intervention Measures

[0051] Patients were randomly assigned to an experimental group and a control group in a 1:1 ratio. The experimental group used LWRF oil topically, while the control group used Yuze Skin Barrier Repair Body Lotion (Shanghai Jahwa United Co., Ltd.). Both were used twice daily for 4 consecutive weeks, followed by a 4-week follow-up.

[0052] Before and after treatment, sterile swabs were used to collect skin flora samples from the neck, elbow, or popliteal fossa of patients in the experimental group, while samples were collected from normal skin areas in the control group.

[0053] 1.3 Outcome Indicators

[0054] The primary outcome was the relapse rate during remission (IGA > 2 points defined as relapse). Secondary outcomes included the Eczema Area and Severity Index (EASI), Investigator Global Assessment (IGA), and Dermatology Quality of Life Index (DLQI). Assessments were performed at baseline (week 0), week 4 of treatment, and week 8 of follow-up.

[0055] Statistical analysis was performed using SPSS 25.0, with p < 0.05 considered statistically significant. Qualitative indicators were described as frequencies, percentages, or proportions; quantitative indicators, when normally distributed, were expressed as mean ± standard deviation, and when not normally distributed, as median and interquartile range. Two-sample t-tests were used for normally distributed measurement data, and nonparametric rank-sum tests were used for non-normally distributed data; χ² tests were used for unordered categorical data, and rank-sum tests were used for ordered categorical data.

[0056] 1.4 Metabolomics Analysis

[0057] The Q300 quantitative metabolomics technique was used to detect changes in skin microbiota metabolites before and after treatment in normal skin controls and AD patients. Prior to sampling, subjects were required to have not used antibiotics at the sampling site for 6 months and had not applied any topical medications or skincare products within 12 hours. During sampling, the skin was not cleaned; under suitable ambient temperature, a sterile sampling swab was used to swab the inner elbow, popliteal fossa, and anterior neck (approximately 4 cm²) 10 times. Samples were transported on dry ice and stored at -80°C.

[0058] Each swab sample was placed in a microcentrifuge tube, and 10 pre-chilled zirconia beads and 120 μl of methanol solution containing internal standard were added to extract metabolites. Samples were homogenized for 3 minutes, vortexed for 5 minutes, and centrifuged at 18000×g for 20 minutes. 20 μl of the supernatant was transferred to a 96-well plate, and 20 μl of freshly prepared derivatization reagent was added to each well. The plate was sealed and derivatized at 30°C for 60 minutes. After derivatization, the sample was allowed to evaporate for 2 hours, then reconstituted with 330 μl of ice-cold 50% methanol solution. The plate was incubated at -20°C for 20 minutes and then centrifuged at 4000×g at 4°C for 30 minutes. 135 μl of the supernatant was transferred to a new 96-well plate (each well containing 10 μl of internal standard), and serially diluted derivatization standards were added to the remaining wells. The plates were sealed and analyzed by LC-MS.

[0059] All metabolites were quantified using an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) system (ACQUITYUPLCXevo TQ-S, Waters Corp.). Chromatographic separations were performed using an ACQUITY UPLC BEH C18 1.7 μM VanGuard pre-column (2.1 × 5 mm) and an ACQUITY UPLC BEH C18 1.7 μM analytical column (2.1 × 100 mm). The mobile phase was water containing 0.1% formic acid and acetonitrile / isopropanol (70:30), with a flow rate of 0.40 mL / min and a sample temperature of 10 °C. Raw data were peak integrated, calibrated, and quantified using TMBQ software (v1.0, Metabo-Profile).

[0060] Metabolomics Quality Control and Reproducibility: Absolute quantification was performed using the targeted metabolomics analysis platform (Metabo-Profile, Shanghai, China), which boasts high reproducibility. Internal standards were added to all samples to monitor analytical variability during preparation and detection. Pooled QC samples were prepared by aliquoting all study samples to represent the overall biological average. QC samples were periodically injected during analysis to assess system stability.

[0061] Metabolite set enrichment analysis: Overrepresentational analysis (ORA) using hypergeometric test was performed to complete the metabolite set enrichment analysis. Holm-corrected p-values ​​and false discovery rate (FDR) were calculated to correct for multiple comparisons. Given the comprehensive coverage of metabolic pathways in the KEGG database, it was selected for pathway enrichment analysis.

[0062] 2. Experimental Results

[0063] 2.1. LWRF oil for preventing recurrence of skin lesions during the remission period of atopic dermatitis

[0064] We conducted a randomized, controlled, single-center clinical trial at Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, affiliated with Shanghai University of Traditional Chinese Medicine, to evaluate the clinical efficacy and safety of LWRF oil in preventing relapse in patients with Alzheimer's disease (AD) during remission. A total of 98 patients were screened, and 96 eligible subjects were randomly assigned to either the intervention group (n=43, topical application of LWRF oil) or the control group (n=42, topical application of Yuze Skin Barrier Repair Body Lotion). The treatment period was 4 weeks, followed by a 4-week follow-up, for a total study duration of 8 weeks. Five patients withdrew due to worsening symptoms, 6 were lost to follow-up, and ultimately 85 patients completed the trial. Figure 3 The demographic and baseline characteristics of the two groups of patients were balanced (Table 2). The primary outcome measure was the relapse rate during remission, and secondary outcomes included the Eczema Area and Severity Index (EASI), Investigator Global Assessment (IGA), Dermatology Quality of Life Index (DLQI), and serum total IgE level.

[0065] In patients in remission of Alzheimer's disease (AD), topical LWRF oil significantly reduced the relapse rate at week 4 (2.3% [1 / 43]; 95% CI: 0.1%–12.1%) and week 8 (16.3% [7 / 43]; 95% CI: 8.1%–30.0%), which was superior to the control group (week 4: 14.3% [6 / 42]; 95% CI: 6.7%–27.8%; week 8: 35.7% [15 / 42]; 95% CI: 23.0%–50.8%). Figure 3 A). Furthermore, the LWRF oil group had significantly lower EASI scores at week 4 (Z = -2.177, p = 0.029, r = -0.24) and week 8 (Z = -1.976, p = 0.048, r = -0.22); within-group analysis showed significant decreases from baseline to both week 4 and week 8. Figure 3 B). The IGA score showed a similar improving trend, with the LWRF oil group showing significantly lower scores at week 4 (Z = -3.026, p = 0.025, r = -0.33) and week 8 (Z = -2.039, p = 0.041, r = -0.22), and a decrease within the group from baseline to both week 4 and week 8. Figure 3 C). DLQI scores in the LWRF oil group were also significantly lower than those in the control group (week 4: Z = -2.105, p = 0.035, r = -0.23; week 8: Z = -2.157, p = 0.031, r = -0.24), with significant decreases from baseline to both week 4 and week 8 within the group. Figure 3 D). Finally, the serum total IgE level in the LWRF oil group was significantly lower than that in the control group at week 4 (Z = -2.001, p = 0.045, r = -0.22), and there was a significant decrease within the group from baseline to week 4. Figure 3 E). No serious adverse events were reported during the 8-week follow-up period. Five patients in the treatment group experienced mild skin erythema, which resolved spontaneously with continued use. In conclusion, topical LWRF oil significantly reduced the recurrence rate and improved clinical outcomes, as evidenced by a decrease in EASI, IGA, DLQI scores and serum IgE levels.

[0066] 2.2. LWRF oil restores skin metabolome by regulating BCAA metabolism.

[0067] To elucidate the mechanism by which LWRF oil prevents AD recurrence, we performed Q300 quantitative metabolomics analysis on swab samples from the skin lesions of AD patients before treatment (AD group, n=9), after treatment (LWRF group, n=9), and from the contralateral healthy skin (HC group, n=9). Principal component analysis (PCA) showed significant separation of metabolites among the groups. Figure 4A): The HC group samples clustered tightly, indicating a consistent metabolic profile; the AD group samples were dispersed along PC1, indicating increased metabolic heterogeneity; after LWRF treatment, the samples converged towards the HC group, suggesting that the skin metabolome was returning to normal. Amino acids were the most abundant metabolite class among all groups. Figure 4 B and Figure 4 C). Compared with the HC group, the amino acid abundance in the AD group was significantly increased, but LWRF treatment reversed this trend. Metabolite enrichment analysis showed that the LWRF group exhibited significant changes in the degradation of branched-chain amino acids (BCAAs; valine, leucine, isoleucine) and the metabolic pathways of phenylalanine and tyrosine. Figure 4 D). Based on the p-value and pathway impact score, the valine, leucine, and isoleucine degradation pathways were selected for in-depth analysis. Figure 4 E). Thermographic visualization confirmed increased BCAA abundance in AD skin, and LWRF oil treatment reversed this phenomenon. Figure 4 F). These results indicate that LWRF oil reduces inflammation by modulating skin metabolism, particularly BCAA degradation.

[0068] 2.3. BCAA accumulation is associated with inflammatory aging of the skin in patients with AD.

[0069] Previous studies have suggested that BCAA accumulation can drive cellular senescence phenotypes, potentially associated with inflammatory senescent skin lesions observed in chronic Alzheimer's disease (AD) patients. To investigate this association, we examined the histopathology and expression of aging markers in the skin tissues of AD patients. Histopathological analysis of skin lesions from AD patients revealed epidermal hyperplasia, cavernous dermatitis, dermal inflammatory cell infiltration, and disordered collagen fiber arrangement, suggesting skin aging. Figure 5 A). Immunohistochemical staining showed that the expression of the aging-related marker P21 in the skin lesions of AD patients was higher than that in healthy controls (n=8 vs n=6); Figure 5 B). Notably, metabolomics analysis showed that skin BCAA levels (valine, leucine, and isoleucine) in AD patients were positively correlated with age. Figure 5 C; Valine: R2 = 0.6839, p = 0.0060; Leucine: R2 = 0.5912, p = 0.0155; Isoleucine: R2 = 0.5237, p = 0.0275). These findings suggest that BCAA accumulation is associated with inflammatory aging in Alzheimer's disease (AD), and targeting skin BCAA metabolism may be a potential strategy to improve inflammatory aging and prevent AD recurrence.

[0070] Example 3 Animal Experiment

[0071] 1. Animal models

[0072] Eight-week-old male BALB / c mice (weighing 22±24g) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed at the Animal Resource Center of Yueyang Hospital of Integrated Traditional and Western Medicine, Shanghai. Mice were housed in a pathogen-free environment (temperature 20-26℃, 12-hour light-dark cycle) with free access to standard feed and water. The animal research protocol was approved by the Laboratory Animal Ethics Committee of Yueyang Hospital of Integrated Traditional and Western Medicine (Approval No.: YYLAC-2023-208-2, Date: August 19, 2024). Mice were randomly divided into four groups (n=5 per group): blank control group, AD model group, LWRF group, and Yuze group. All treatment groups received prophylactic administration starting from day 1. Calcipotriol (MC903, MedChemExpress) was used to induce AD-like skin lesions in mice. MC903 modeling began on day 3 and was performed concurrently with prophylactic treatment. 20μl of 50μM MC903 was applied topically to the right ear of each mouse in each group, while the control group used 10μl of 100% ethanol as a solvent control. The experiment lasted 21 days.

[0073] Mice were weighed and the severity of Alzheimer's disease (AD) was assessed on days 1, 6, 10, 16, and 21. The SCORAD clinical scoring method was as described previously (Wang et al., 2024). Ear symptom scores were calculated after macroscopic assessment of the relative severity of dermatitis. Serum was collected from mice anesthetized with isoflurane after observation, and skin samples were collected after euthanasia.

[0074] 2. BCAT1 activation verification:

[0075] Western blot analysis was performed on the expression levels of BCAT1 and BCAT2 in skin tissues, following the methods described in previous studies (Wang et al., 2024). Samples were detected using primary antibodies against GADPH (1:10000, Proteintech), P21 (1:500, Servicebio), BCAT1 (1:2000, Proteintech), and BCAT2 (1:1000, Proteintech).

[0076] Quantitative analysis of skin using LC-MS / MS (ultra-high performance liquid chromatography-tandem mass spectrometry)

[0077] BCAA levels (methods are described in document 3), with samples obtained from skin swabs of AD patients (n=9) and mouse models.

[0078] 3. Assessment of inflammatory aging:

[0079] Skin samples were fixed in 10% formalin, dehydrated with ethanol of varying concentrations (70%-100%), and embedded in paraffin. Tissue sections (6 μm) were dried overnight at 42°C for later use. Hematoxylin-eosin (H&E) staining: hematoxylin staining for 5 minutes, eosin staining for 5 minutes, and rinsing with acidic alcohol solution. Immunohistochemistry (IHC) was performed according to previous studies (Chen et al., 2022), using anti-P21 antibody (1:500, Servicebio) as the primary antibody.

[0080] The levels of SASP factors (IL-8 and IL-11) in mouse serum cells were measured by ELISA using the commercially available Bio-Plex Pro Mouse Cytokine 23-plex Assay Kit (Bio-Rad).

[0081] 4. Pharmacodynamic analysis:

[0082] Ear tissue samples were digested enzymatically (overnight at 37°C) to prepare single-cell suspensions. The cell suspensions were washed twice with pre-chilled PBS and resuspended in PBS (concentration 1×10⁻⁶). 7 cells / mL). Wash and resuspend again with PBS (1×10⁻⁶ cells / mL) before surface staining. 6(cells / mL). Flow cytometry blank controls were set up (unstained cells, single-stained control, and FMO control). Test samples were incubated with 2 μL of Fc blocking agent at room temperature in the dark for 15 minutes, resuspended in PBS, and then incubated with active dye (1 μL) in the dark for 15 minutes, followed by centrifugation at 500×g for 5 minutes. After washing with 1 mL of Stain Buffer (BD Biosciences, 554656), the samples were resuspended in 200 μL of Stain Buffer. Surface labeling staining: Fluorescently labeled antibody was incubated at room temperature in the dark for 15 minutes, washed with Stain Buffer, and intracellular cytokine staining was performed using the BD Cytofix / Cytoperm Kit (554714). After fixation / permeabilization, the samples were washed twice with Perm / Wash Buffer, resuspended in 200 μL of Stain Buffer, and stained with intracellular targets at 4°C in the dark for 30 minutes. After washing with Stain Buffer, the samples were resuspended in 400 μL of Stain Buffer and analyzed within 3 hours (LSRFortessaX-20, BD Biosciences). Antibodies used: APC-CyTM7 Rat Anti-Mouse CD45 (557659), FITC Hamster Anti-Mouse CD3e (553061), BB700Rat Anti-Mouse CD4 (566408), and PE-CyTM7 Mouse Anti-GATA3 (560405). Data were analyzed using FlowJo software (v10.8.1).

[0083] 5. Experimental Results

[0084] (1) Selective activation of BCAT1 and metabolic regulation of BCAA

[0085] In an MC903-induced AD mouse model, LWRF Oil treatment significantly upregulated BCAT1 expression, while BCAT2 was unaffected. Figure 6 D). Western blot results showed that BCAT1 protein levels were increased in the LWRF group compared to the MC903 group (p<0.001), which directly promoted BCAA degradation. Skin BCAA levels (leucine, valine, isoleucine) were decreased (p<0.01). Figure 6 A), consistent with clinical AD patient data: BCAA levels returned to the level of healthy controls after treatment. Figure 4 F).

[0086] In AD patients, BCAA accumulation was positively correlated with age (R² = 0.52–0.68, p < 0.05), indicating that BCAA drives inflammatory aging. Figure 5 C).

[0087] (2) Reversal of inflammatory aging

[0088] LWRF Oil significantly reduced the aging markers P21 and SASP. IHC showed a 60% reduction in P21 expression (p<0.001). Figure 6 C), and IL-8 and IL-11 levels decreased by 50-65% (p<0.01). Figure 6 B). This confirms that BCAT1 activation can reverse the skin aging microenvironment.

[0089] (3) Th2 cell regulation and clinical efficacy

[0090] LWRF Oil inhibited Th2 cell infiltration, and flow cytometry showed a 55% reduction in the proportion of GATA3+ cells (p<0.001). Figure 7 E). In clinical trials, the relapse rate in AD patients decreased to 16.3% (vs. 35.7% in control), and EASI and IGA scores significantly improved. Figure 3 B and Figure 3 C).

[0091] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of the composition in the preparation of products for the prevention and / or treatment of diseases caused by overexpression of branched-chain amino acids BCAA, wherein: The composition comprises the following components in parts by weight: 20-40 parts of hemp seed, 20-40 parts of angelica, 5-15 parts of notoginseng, 10-30 parts of chuanxiong, 20-40 parts of coix seed, and 20-40 parts of licorice.

2. The application according to claim 1, characterized in that, The composition comprises the following components in parts by weight: 30 parts hemp seed, 30 parts angelica root, 10 parts Panax notoginseng, 15 parts Ligusticum chuanxiong, 30 parts coix seed, and 30 parts licorice root.

3. The application according to claim 1 or 2, characterized in that, The diseases mentioned include at least one of skin diseases, metabolic diseases, nervous system diseases, and age-related diseases.

4. The application according to claim 3, characterized in that, The skin diseases mentioned include Th2 cell-mediated skin diseases.

5. The application according to claim 3 or 4, characterized in that, The disease is selected from at least one of atopic dermatitis, senile pruritus, photoaging, psoriasis, chronic eczema, type 2 diabetes, obesity, non-alcoholic fatty liver disease, hepatic encephalopathy, maple syrup diabetes, frailty syndrome, and cognitive decline.

6. The application according to claim 1 or 2, characterized in that, The composition upregulates the expression of branched-chain aminotransferase 1.

7. The application according to claim 1 or 2, characterized in that, The composition inhibits the expression of the aging marker P21.

8. The application according to claim 1 or 2, characterized in that, The composition inhibits the expression of the aging-related secretory phenotype factor SASP.

9. The application according to claims 1 to 8, characterized in that, The products include pharmaceuticals and / or cosmetic products.

10. The application according to claim 9, characterized in that, The product is any one of emulsion, oil, gel, cream, lotion, spray, sustained-release formulation, or controlled-release formulation.

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