Application of betulinic acid 28-O-beta-D-glucoside in preparation of medicine for preventing and treating skin inflammation

Abstract

The invention discloses an application of betulinic acid 28-O-beta-D-glucoside (BA-6 for short) in preparation of a medicine for preventing and treating skin inflammation. The problems that an existing medicine for treating dermatitis is limited in curative effect, slow in effect taking, multiple in side effect, high in long-term use risk, large in individual difference, high in treatment cost, likely to cause immunoreaction and the like are solved. The invention discloses application of BA-6 in prevention and treatment of atopic dermatitis, contact dermatitis, eczema, rash, psoriasis, lupus erythematosus, papule squamous skin disease, pityriasis rosea, seborrheic dermatitis, pemphigus, celestial abscess, acne, tinea corporis, impetigo, SAPHO syndrome, hidradenitis suppurativa, dermatitis caused by drugs, lichen planus, pityriasis rubra pilaris, vitiligo, prurigo nodularis, dermatitis caused by drugs, and the like for the first time. The traditional Chinese medicine composition can be used for treating skin diseases, systemic sclerosis, hidradenitis suppurativa, granulomatous skin diseases, vitiligo, dermatomyositis, chronic skin itch and alopecia areata, and has the technical progress of obviously improving skin erythema, papule, edema, scale, moss and ulceration symptoms.

Description

[0001] This application is a divisional application of the application dated May 27, 2025, application number “2025106909088”, entitled “Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs for the prevention and treatment of skin inflammation”. Technical Field

[0002] This invention belongs to the field of pharmacology of natural products, specifically relating to the application of betulinic acid derivative - betulinic acid 28-O-β-D-glucoside (BA-6) in the preparation of drugs for treating or preventing skin inflammation. Background Technology

[0003] Inflammatory skin diseases are a group of skin inflammatory reactions caused by multiple factors. Their pathogenesis is complex and may involve intrinsic factors such as the skin microbiome, genetics, abnormal immune function, mental disorders, and lifestyle factors (including diet, smoking, alcohol consumption, and sleep patterns). Furthermore, stimulation of the skin by pathogens in the external environment can also trigger an inflammatory response. From a modern medical perspective, inflammatory skin diseases are mainly classified into five categories: infectious inflammatory skin diseases, immune-related inflammatory skin diseases, allergic inflammatory skin diseases, non-infectious inflammatory skin diseases, and other inflammatory skin diseases. Common inflammatory skin diseases include psoriasis, atopic dermatitis, chronic urticaria, eczema, vitiligo, acne, and rosacea. These diseases present with varying clinical manifestations, and their pathogenesis is not fully understood, but they are recognized to share common characteristics: inflammatory and immune factors are involved in their pathogenesis, including skin inflammatory responses mediated by T cells and antigen-antibody interactions. Drug treatment for inflammatory skin diseases mainly includes topical medications and systemic therapies. Topical medications include corticosteroids, calcineurin inhibitors, vitamin D3 derivatives, retinoic acid, and antibiotics; systemic medications include antihistamines, corticosteroids, traditional immunosuppressants, and antibiotics. Phototherapy is also a commonly used treatment method. In recent years, with in-depth research into the pathogenesis of inflammatory skin diseases, new treatment methods such as biologics and small-molecule targeted drugs have emerged, bringing more choices and hope to patients.

[0004] Atopic dermatitis (AD) is a relapsing, chronic, non-infectious inflammatory skin disease. AD typically presents with itchy, erythematous plaques, crusting, cracking, and lichenification, often accompanied by seasonal fluctuations. The distribution of lesions differs between children and adults; it usually develops in childhood and may persist into adulthood. According to the Global Burden of Disease study, the prevalence in children is 15% to 20%, while the prevalence in adults is as high as 10%, making atopic dermatitis the 15th most common non-fatal disease and the skin disease with the highest disease burden, causing significant physical, psychological, and socioeconomic burdens. The interaction between genetic and environmental factors, skin barrier dysfunction, microbial imbalance, immune dysregulation, and environmental triggers of skin inflammation play a role in the pathogenesis of atopic dermatitis. Inflammation is thought to be triggered by disruption of the epidermal barrier and activation of epidermal dendritic and innate lymphocytes, which attract and interact with invading Th2 cells. The direct mechanism of eczema lesions is inflammation associated with Th2 cell dysregulation. Activated T cells release cytokines into the skin, primarily interleukin-4, interleukin-13, and interleukin-31, which activate the downstream Janus kinase (JAK) pathway. Cytokines promote inflammation, pruritus, and antigen-specific IgE production by activating B cells and plasma cells. Pruritus in atopic dermatitis is based on signal transduction between prurigotropic agents released by keratinocytes, mast cells, and immune cells (T cells and eosinophils) and small sensory nerve fibers in the skin. Prurigotropic agents include Th2 cytokines (especially interleukin-4, -13, and -31), thymic stromal lymphopoietin (an epithelial-derived pro-inflammatory cytokine), histamine, proteases, and neuropeptides. These prurigotropic agents bind to receptors on sensory C and Aδ nerve fibers in the epidermis and dermis, which sense itching and pain.

[0005] Currently, treatment for atopic dermatitis is chosen based on the clinical stage of the disease (mild, moderate, or severe), the extent of affected body surface area, age, coexisting diseases and medications, severity of itching, degree of impairment in quality of life, and patient goals. Treatment primarily includes topical and oral medications. Mild atopic dermatitis mainly uses topical medications, including corticosteroids (TCS), calcineurin inhibitors (TCI), phosphodiesterase 4 inhibitors (PDE-4 inhibitors), and Janus kinase inhibitors (JAK inhibitors). Glucocorticoids (TCS), as first-line treatment, can rapidly control the condition, but long-term, large-area use is not recommended. Prolonged use may cause adverse reactions such as skin atrophy, barrier dysfunction, and even steroid-dependent dermatitis. Calcineurin inhibitors (TCI) and phosphodiesterase 4 inhibitors (PDE-4 inhibitors) may cause local burning, stinging, or increased itching in some patients, which may affect medication adherence. Moderate to severe atopic dermatitis requires combination therapy, including antihistamines, immunosuppressants such as cyclosporine, azathioprine, and methotrexate, systemic corticosteroids such as prednisone, and biologics such as dupilumab and trororutumab. Long-term use of corticosteroids can cause metabolic disorders and obesity, and in some patients, hypertension, increasing the risk of cardiovascular disease. First-generation antihistamines such as diphenhydramine and chlorpheniramine have strong central nervous system depressant effects, and some antihistamines such as astemizole and terfenadine may cause cardiotoxicity. Biologics are generally expensive, and long-term use can impose a significant financial burden on patients. Biologics control inflammation by modulating the immune system, but may also increase the risk of infection. Some patients may experience allergic reactions such as rash, itching, and difficulty breathing, which can be life-threatening in severe cases. JAK inhibitors regulate the immune system by inhibiting the JAK-STAT signaling pathway, but may also increase the risk of infection, especially upper respiratory tract infections and herpes zoster. JAK inhibitors may cause abnormal blood counts. Since the pathogenesis of Alzheimer's disease (AD) is not yet clear, and there is currently no cure with medication and no specific drugs, clinical treatment focuses more on controlling the progression of the disease and improving the patient's quality of life. From the perspective of overall application effects, dexamethasone is currently the main treatment for AD in clinical practice. Summary of the Invention

[0006] In view of the problems of existing drugs for treating dermatitis, such as limited efficacy, slow onset of action, many side effects, high risks of long-term use, large individual differences, high treatment costs, and possible immune reactions, this invention discloses for the first time the application of betulinic acid 28-O-β-D-glucoside (also known as betulinic acid 28-O-β-D-glucopyranoside, abbreviated as BA-6) in the prevention and treatment of dermatitis, which has a significant technological advancement in improving dermatitis symptoms.

[0007] The present invention adopts the following technical solution.

[0008] Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs for the prevention and treatment of skin inflammation.

[0009] Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs for the prevention and treatment of skin erythema and papules.

[0010] Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs for the prevention and treatment of pruritus.

[0011] Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs to reduce skin edema and epidermal thickening.

[0012] Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs to reduce skin scaling.

[0013] Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs to reduce skin lichenification.

[0014] Application of betulinic acid 28-O-β-D-glucoside in the preparation of drugs for the prevention and treatment of skin ulcers.

[0015] In this invention, skin inflammation includes atopic dermatitis, contact dermatitis, eczema, paronychia, rashes, psoriasis, lupus erythematosus, papulosquamous dermatitis, pityriasis rosea, seborrheic dermatitis, pemphigus, pustular dermatitis, acne, tinea corporis, impetigo, SAPHO syndrome, hidradenitis suppurativa, neurodermatitis, drug-induced dermatitis, lichen planus, pityriasis rubra pilaris, vitiligo, nodular prurigo, systemic sclerosis, bedsores, granulomatous dermatitis, dermatomyositis, urticaria, chronic pruritus, or alopecia areata; preferably, skin inflammation includes atopic dermatitis, contact dermatitis, eczema, rashes, psoriasis, lupus erythematosus, papulosquamous dermatitis, pityriasis rosea, seborrheic dermatitis, vitiligo, acne, and tinea corporis. The betulinic acid 28-O-β-D-glucoside disclosed in this invention can not only treat or prevent dermatitis and its symptoms, but also has the advantages of low cytotoxicity and safety.

[0016] This invention discloses a pharmaceutical composition for treating skin inflammation, with betulinic acid 28-O-β-D-glucoside as the active ingredient; it may also include conventional pharmaceutical excipients, including one or more of diluents, dispersants, binders, lubricants, and penetration enhancers.

[0017] Specifically, conventional pharmaceutical excipients refer to one or more compatible solid or liquid fillers or gel substances that are pharmaceutically usable, have sufficient purity and low toxicity, and can be mixed with other components in the pharmaceutical composition and with the active ingredient of the present invention without reducing the efficacy of the active ingredient. Pharmaceutically acceptable carriers include, in part, cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), cyclodextrins (such as hydroxypropyl β-cyclodextrin), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0018] In this invention, the dosage forms of the drug include pills, tablets, powders, pastes, ointments, granules, capsules, nano-preparations, aerosols, sprays, ointments, solutions, injections, sustained-release preparations, controlled-release preparations, gels, or suppositories.

[0019] In this invention, the drug or drug composition includes topical, oral, rectal, or parenteral drugs. Preferably, the drug is a solution.

[0020] Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid compound found in various plant species, such as *Pulsatilla chinensis* (Ranunculaceae), *Syzygium buergerianum* (Myrtaceae), and *Nerium oleander*. It can also be prepared from its metabolic precursor, betulinol. BA and its derivatives (synthesized through modifications at C-3, C-20, and C-28 sites) possess a variety of biological activities, including anticancer, anti-HIV, antiparasitic, and anti-angiogenic activities. In particular, BA has been reported to have cytotoxic effects against several tumor cell lines from different sources and in animal models of cancer. While BA exhibits potent anti-HIV and antitumor activities, it is considered to possess relatively weak anti-inflammatory activity. According to relevant research reports, in lipopolysaccharide (LPS)-induced endotoxin shock, BA pretreatment exhibits anti-inflammatory activity through the L-10 mechanism, but its anti-inflammatory activity and protective effect are weaker than dexamethasone. BA derivative BA5 (chemical structural name: 3β-hydroxy-lupinane-20(29)-ene-28-acyl-morpholine) protects mice from lethal LPS attack and reduces edema in delayed-type hypersensitivity reactions, but its effect is also weaker than dexamethasone. The betulinic acid 28-O-β-D-glucoside (BA-6) designed and synthesized in this invention is a glycoside derivative of BA. Its water solubility and bioavailability are significantly improved compared to BA. In particular, BA-6 has a significant effect on improving dermatitis, and its efficacy is significantly better than dexamethasone, which is beyond the expectations of those skilled in the art. Attached Figure Description

[0021] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0022] Figure 1 This diagram illustrates the method for establishing a mouse model of DNCB-induced dermatitis.

[0023] Figure 2 Statistical graph showing the effect of BA-6 on body weight (g) in mice with DNCB-induced dermatitis; compared with the normal control group, ##P<0.01, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the dexamethasone group (DEX, 3 mg / kg), &P<0.05, &&P<0.01; n≥7.

[0024] Figure 3 The graph shows the effect of betulinic acid 28-O-β-D-glucoside on the dorsal skin of mice with DNCB-induced dermatitis and the statistical results of EASI scores. A represents the dorsal skin of mice with dermatitis, and B represents the statistical results of EASI scores. Compared with the normal control group, ###P<0.001; compared with the model group, *P<0.05, ***P<0.001; compared with the dexamethasone group (DEX, 3 mg / kg), &P<0.05, &&P<0.01, &&&P<0.001; n≥7.

[0025] Figure 4 The following are statistical graphs showing the effects of BA-6 on ear skin in mice with DNCB-induced atopic dermatitis, as well as the differences in ear thickness and ear weight. A represents the ear skin of mice with dermatitis, B represents the differences in ear thickness, and C represents the differences in ear weight. Compared with the normal control group, ### P<0.001; compared with the model group, *** P<0.001; compared with the dexamethasone group (DEX, 3 mg / kg), & P<0.05, && P<0.01, &&& P<0.001; n≥7.

[0026] Figure 5 Statistical graph showing the effect of BA-6 on spleen index in mice with DNCB-induced atopic dermatitis; compared with the normal control group, ### P<0.001; compared with the model group, ** P<0.01, *** P<0.001; compared with the dexamethasone group (DEX, 3mg / kg), &&& P<0.001; n≥7.

[0027] Figure 6The effect of BA-6 on serum inflammatory factors FN-γ, IL-6, and IL-1β in mice with DNCB-induced atopic dermatitis was investigated, where A represents IFN-γ, B represents IL-6, and C represents IL-1β. Compared with the normal control group, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001; compared with the dexamethasone group (DEX, 3 mg / kg), & P<0.05; n=3.

[0028] Figure 7 This is a statistical graph showing the survival rate of BA-6 in mice with DNCB-induced atopic dermatitis.

[0029] Figure 8 This study used an ELISA assay to evaluate the effects of BA-6 and betulinic acid (BA) on the release of LPS-induced inflammatory factors IL-1β, IL-6, and TNF-α. In the assay, A represented IL-1β, B represented IL-6, and C represented TNF-α. Compared with the normal control group, ### P < 0.001; compared with the model group, *P < 0.001, ***P < 0.001; compared with the betulinic acid (BA, 10 μM) group, $$P < 0.01, $$$P < 0.001; n = 3.

[0030] Figure 9 This is a CCK-8 cytotoxicity assay; cell viability greater than 90% is generally considered non-toxic or low-toxicity; A represents the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) and betulinic acid (BA) at a concentration of 50 μM on THP-1 cells induced to differentiate into M1 macrophages; B represents the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) and betulinic acid (BA) at a concentration of 50 μM on RAW264.7 cells; C represents the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) at concentrations of 50, 100, 150, 200, 250, and 300 μM on THP-1 cells induced to differentiate into M1 macrophages; D represents the IC50 value of betulinic acid 28-O-β-D-glucopyranoside (BA-6), IC50 = 261.5 μM; n = 3. Detailed Implementation

[0031] The betulinic acid 28-O-β-D-glucoside (BA-6) disclosed in this invention can not only treat or prevent dermatitis and its symptoms, but also has low cytotoxicity.

[0032] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the data analysis is performed using conventional statistical analysis methods. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the reagents and materials are commercially available, and the animal experiments meet the relevant requirements of Soochow University.

[0033] In this invention, the chemical structures of betulinic acid 28-O-β-D-glucoside (compound BA-6) and betulinic acid (compound BA) are as follows:

[0034]

[0035] Betulinic acid 28-O-β-D-glucoside can be obtained by conventional methods. An example of this invention is as follows:

[0036]

[0037] Add 125 mg of iodine to 25 mL of acetic anhydride solution containing 5.00 g of D-pyranose. Stir magnetically at room temperature until the reaction mixture turns brown and transparent. After the reaction is complete, dilute the reaction mixture with 125 mL of dry dichloromethane. Cool in an ice bath and add 30 mL of glacial acetic acid solution containing 33% hydrogen bromide. After the addition is complete, stir the reaction mixture at room temperature until TLC detection shows that the reaction is complete. Dilute the reaction mixture with 125 mL of dichloromethane and wash successively with ice water, saturated sodium bicarbonate solution, and saturated sodium thiosulfate solution.

[0038] The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a colorless oily substance. Recrystallization from diethyl ether and petroleum ether yielded a white solid powder (G3) with a yield of 95%. Immediately, BA (3 g, 6.569 mmol) and G3 (3.5 g, 8.539 mmol) were dissolved in a CH2Cl2 / H2O (76 ml / 76 ml) mixture. n-Bu4NBr (847 mg, 2.628 mmol) and K2CO3 (2.2 g, 16.421 mmol) were added sequentially, and the mixture was stirred at room temperature for 6 h. The mixture was then diluted with 100 ml of dichloromethane, and the organic phase was washed with saturated brine (100 mL × 3). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 → 3:1) to give 4.3 g of a white solid (G4) with a yield of 83%. G4 (4 g, 5.083 mmol) was dissolved in a THF / H2O (100 ml / 10 ml) mixture, and sodium hydroxide (1.2 g, 30.498 mmol) was added. The mixture was stirred at room temperature for 12 h, and then 1 M HCl was added to adjust the pH to 4. Water was then added to precipitate the product, which was filtered, dried, and reconstituted with methanol. The product was then separated by C18 column chromatography (mobile phase: 90% methanol-water) to obtain 2.5 g of betulinic acid-28-O-β-D-glucoside (BA-6) as a white solid, with a yield of 79%. NMR and MS data are as follows:

[0039] 1H NMR (400 MHz, CD3OD) δ 5.49 (1H, d, J = 8.2 Hz, H-1'), 4.72 (brs,H1-29), 4.60 (brs,H2-29), 3.84 (1H, dd, J = 12.0, 1.6 Hz), 3.73-3.68 (1H, m),3.45-3.39 (1H, m), 3.39 – 3.36(2H, m), 3.35 (2H, m), 3.12 (1H, dd, J = 11.1,5.0 Hz), 3.01 (1H, m), 2.33 (2H, m), 2.02-1.88(2H, m), 1.75-1.71 (1H, m),1.70 (3H, s), 1.69-1.19 (17H, m), 1.16 (1H, m), 1.05 (1H, m),1.00 (3H, s),0.96 (3H, s), 0.94 (3H, s), 0.86 (3H, s), 0.75 (3H, s), 0.71 (1H, m). 13C NMR (101MHz, (CD3OD) δ 176.1 (C-28), 151.8 (C-20), 110.3 (C-29), 95.2 (C-1'), 79.7 (C-3), 78.8 (C-5'), 78.4 (C-3'), 74.1 (C-2'), 71.1 (C-4'), 62.4 (C-6'), 57.9, 56.9,52.0, 50.6, 49.9, 43.5, 42.0, 40.1, 39.9, 39.4, 38.3, 37.5, 35.5, 32.8, 31.4,30.8, 28.6, 28.0, 26.8, 22.1,19.5, 19.4, 16.7, 16.6, 16.1, 15.1. HR-MS m / zcalcd for C 36 H 58 O8K [M + K] + 657.3763, found657.3738. HPLC purity: 99.12%.

[0040] Example 1: Method for establishing a mouse model of (atopic) dermatitis induced by DNCB

[0041] BALB / c mice, SPF grade, female, 7-8 weeks old, weighing 18-21g, were acclimatized for one week and then weighed and randomly divided into eight groups: normal control group, model group, positive drug dexamethasone control group (DEX, 3 mg / kg), BA-6 (2.5 mg / kg), BA-6 (5 mg / kg), and BA-6 (10 mg / kg).

[0042] Preparation of back and ear modeling agents: Using acetone:olite = 4:1 as solvent and DNCB as solute, prepare 5% DNCB back modeling agent and 1% DNCB ear modeling agent respectively. Preparation of drug solution: Weigh 60 mg of BA-6 and prepare a solution with ethanol:water:glycerol = (70:28:2, volume ratio) to a concentration of 1 mg / mL. Other dosages are prepared according to the proportions.

[0043] Two days prior to the experiment, the backs of mice were shaved using a shaver, covering an area of ​​approximately 3cm × 3cm. Then, depilatory cream was used to remove the vellus hair from the backs. On Day 1, the sensitization phase, except for the normal control group, at 2 PM, 50 μL of 5% DNCB solution was evenly applied to the backs of all mice in all other groups to sensitize the skin. The normal control group received only the solvent. On Day 2, the procedure from Day 1 was repeated to re-sensitize the backs of the mice. The normal control group received only the solvent. On Day 3, the challenge phase, except for the normal group, at 2 PM, 100 μL of 1% DNCB solution was applied to the right ear of all mice in all other groups for challenge, with 50 μL applied to each of the inner and outer sides of the right ear. On Days 4 and 5, the procedure from Day 3 was repeated, and challenge was continued in the right ears of the mice for three days to induce and establish a mouse model of atopic dermatitis. The successful establishment of the model was demonstrated by the appearance of redness, swelling, ulceration, exudation, papules, desquamation, and hardening of the skin on the back of mice under repeated stimulation with DNCB solution; and redness, erosion, and hardening of the skin on the ears. Administration method: Mice in the dexamethasone (DEX, 3 mg / kg) group were given dexamethasone cream on their backs and the inner and outer sides of their right ears at 4 pm every day (1-7 days); Mice given BA-6 (2.5 mg / kg), BA-6 (5 mg / kg), and BA-6 (10 mg / kg) groups were given a drug-containing solution on their backs and the inner and outer sides of their right ears at 10 am and 4 pm every day (1-7 days).

[0044] The normal control group and the model group were treated with equal amounts of a mixed solvent of ethanol:water:glycerol (70:28:2) on the back and inner and outer sides of the right ear of each mouse at the same time intervals as the drug-treated groups, maintaining consistency with the overall behavior of the drug-treated groups. On day 8, the thickness of the left and right ears of each mouse was measured, and the mice were sacrificed. The spleen and ears were harvested, and the spleen index, ear weight, and ear thickness difference were calculated (ear discs were obtained by punching the same area with a 6 mm diameter punch and weighing them). The experimental procedure is as follows: Figure 1As shown.

[0045] Example 2: Effect of betulinic acid 28-O-β-D-glucoside on body weight in DNCB-induced atopic dermatitis mice

[0046] The daily changes in body weight of each group of mice were statistically analyzed and plotted using Graph Prism 8.3.0, as shown below. Figure 2 As shown, the weight of mice in the normal control group remained stable, while the weight of mice in the DNCB-induced model group decreased sharply from day 3 of modeling, with a significant difference compared to the normal control group from day 3 to day 6 (P<0.01), consistent with the symptoms of DNCB-induced atopic dermatitis in mice. Compared with the model group, the BA-6 (2.5 mg / kg) group showed a significant difference on day 5 (P<0.05), while the dexamethasone group showed no significant difference. Compared with the dexamethasone group, the BA-6 (2.5 mg / kg) group showed significant differences on days 5 and 8 (P<0.05), and the BA-6 (5 mg / kg) and BA-6 (10 mg / kg) groups showed significant differences on day 8 (P<0.05). Therefore, BA-6 improves the trend of weight loss in DNCB-induced atopic dermatitis mice, and its efficacy is superior to dexamethasone.

[0047] Example 3: Effects of betulinic acid 28-O-β-D-glucoside on the size, severity, and overall assessment score (EASI) of dorsal skin lesions in DNCB-induced atopic dermatitis mice.

[0048] When local skin tissue comes into contact with DNCB, DNCB binds to proteins on the surface of skin cells to form a hapten, activating the immune system and ultimately leading to typical symptoms such as redness, swelling, itching, exudation, ulceration, and telangiectasia. The EASI scoring system is as follows: It consists of four symptom indicators: erythema, papules / edema, scaling, and lichenification / crusting, as shown in Table 1. Each indicator is scored from 0 to 3 points according to its severity, with 0 points for no symptoms, 1 point for mild symptoms, 2 points for moderate symptoms, and 3 points for severe symptoms. All scores are summed to obtain the total score, with a maximum total score of 12 points.

[0049] like Figure 3 As shown, during the experiment, the dorsal skin of mice in each group was observed and photographed on days 1, 3, 5, and 7. It was found that from day 2 of modeling, erythema and papules began to appear on the dorsal skin of DNCB mice, accompanied by signs of ulceration. With subsequent repeated sensitization and ear provocation, the dorsal skin of the model group mice showed obvious redness, swelling, crusting, hardening, and lichenification lesions, while the dorsal skin of the normal group mice remained smooth and intact throughout. Compared with the model group, the dorsal skin of the BA-6-treated group mice showed...

[0050] During the same period, the skin damage symptoms were all alleviated, with no obvious redness, swelling, exudation, or scaling. Erythema, papules, and skin hardening were significantly reduced. On day 5, signs of scab formation and new skin regeneration appeared. In the dexamethasone-treated group, the redness and swelling on the back of the mice were reduced on day 3, but later, similar ulceration, increased hardening, and increased redness and swelling appeared as in the model group. Compared with the normal control group, the EASI scores on the back of the mice in the model group were statistically significant on days 3, 5, and 7 (P<0.001). Compared with the model group, the EASI scores on the back of the mice in the BA-6-treated group increased slowly from day 3, which was statistically significant (P<0.001). The EASI scores on the back of the mice in the dexamethasone group were lower than those in the model group on day 3 (P<0.05), but there was no difference between them on days 5 and 7. Compared with the dexamethasone group (DEX), the BA-6-treated group showed better improvement on the back of the mice, with statistically significant EASI scores on days 3, 5, and 7 (P<0.05). The above data indicate that BA-6 has a superior therapeutic effect on the dorsal skin of AD mice compared to dexamethasone.

[0051] Table 1 EASI Scores

[0052] erythema papules / edema Scales Lichenification / Crusting score none none none none 0 Mild Mild Mild Mild 1 moderate moderate moderate moderate 2 Severe Severe Severe Severe 3

[0053] Example 4: Effects of BA-6 on the size and severity of lesions in the ear skin of mice with DNCB-induced atopic dermatitis, as well as ear thickness and ear weight difference.

[0054] On day 8, photographs were taken of the left and right ears of mice in each group. The thickness of the left and right ears was measured using calipers (measurements were taken from the same location on each ear to minimize error). The ear thickness difference was calculated using the formula: Ear thickness difference = Right ear thickness - Left ear thickness. After euthanizing the mice on day 8, the ears were removed. The two ears were overlapped, and a 6 mm diameter punch was used to create ear discs at the same location. The left and right ear discs were weighed separately. The ear weight difference was calculated using the formula: Ear weight difference = Right ear weight - Left ear weight. Figure 4 As shown, the right ear of the normal group mice was smooth with clear blood vessels and no redness or crusting. Compared with the normal control group, the right ear of the model group mice was red, swollen, and crusted. The ear thickness and ear weight difference of the model group mice were significantly larger than those of the normal control group (P<0.001), indicating that the ear edema and thickening of the model group mice were severe. Compared with the model group, the ear thickness and ear weight difference of all treatment groups were reduced, which was statistically significant (P<0.001). Among them, compared with the dexamethasone group, BA-6 had a more significant effect in reducing ear weight and ear thickness difference in mice, which was statistically significant (P<0.05). The above data show that BA-6 can significantly improve DNCB-induced ear swelling in mice and reduce ear weight and ear thickness difference, and its efficacy is better than that of dexamethasone.

[0055] Example 5: Effect of BA-6 on spleen index in DNCB-induced atopic dermatitis mice

[0056] DNCB-induced skin inflammation primarily activates Th2 cells and may also activate the systemic immune system, leading to increased immune cell activity in the spleen.

[0057] Cell proliferation and activation lead to an increase in spleen volume. For example... Figure 5 As shown, compared with the normal group, the spleen index of the model group mice was significantly increased (P<0.001); compared with the model group, the spleen index of the BA-6 administration group was decreased, with the BA-6 (5 mg / kg) administration group showing a statistically significant difference compared with the model group (P<0.01). The spleen index of the dexamethasone group was extremely low, even significantly lower than that of the normal control group (P<0.001); compared with the spleen index of the dexamethasone group, the spleen index of the BA-6 administration group was also statistically significant (P<0.001). This indicates that dexamethasone induced immunosuppression in mice, while BA-6 had an immunomodulatory effect without immunosuppression, suggesting that the drug had a positive regulatory effect on the pathological state of the spleen or immune function, improving the immunity of mice under stress, inflammation, and other stimuli.

[0058] Example 6: Effects of BA-6 on inflammatory factors in the serum of mice with dermatitis

[0059] On day 8, all mice were euthanized by cervical dislocation after blood was collected from their eyeballs. Whole blood was allowed to settle naturally at 4°C for 2 hours, then centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant serum was collected, and the expression of inflammatory factors IFN-γ, IL-6, and IL-1β in the serum was detected using ELISA according to the kit instructions. Figure 6 As shown, compared with the normal control group, the serum levels of IFN-γ, IL-6, and IL-1β in mice with DNCB-induced atopic dermatitis were significantly increased; compared with the model group, the serum levels of inflammatory factors in the treatment groups were decreased; among them, the BA-6 (5 mg / kg) treatment group showed a more significant decrease in IL-6 compared with dexamethasone, and the difference was statistically significant (P<0.05). These data indicate that BA-6 improves DNCB-induced atopic dermatitis in mice by reducing serum inflammatory factors.

[0060] Example 7: Effects of BA-6 on survival rate and behavior in mice with DNCB-induced atopic dermatitis

[0061] The number of surviving BABL / c mice in each group was statistically analyzed daily and plotted using Graph Prism 8.3.0. (See figure.) Figure 7As shown, the survival rates of the normal control group, model group, dexamethasone (DEX, 3 mg / kg) group, BA-6 (2.5 mg / kg) group, BA-6 (5 mg / kg) group, and BA-6 (10 mg / kg) group were 100%, 88.9%, 77.8%, 100%, 100%, and 100%, respectively. The results indicate that excessive immunosuppression in the dexamethasone group exacerbated mortality in mice with atopic dermatitis, while BA-6 had a protective effect, inhibiting DNCB-induced mortality in mice with acute atopic dermatitis. Scratching behavior was significantly increased in the model group, while it was significantly reduced in the BA-6 and dexamethasone groups, especially in the BA-6 dosage groups where scratching behavior was less than in the dexamethasone group.

[0062] Example 8: In vitro anti-inflammatory experiment of BA-6 and betulinic acid

[0063] THP-1 cells were used at a rate of 5 × 10⁻⁶ 5 THP-1 cells were seeded at a density of cells / well in 24-well plates. Phlorbol-12-Myristate-13-Acetate (PMA) at a concentration of 100 ng / mL was added to induce cell adhesion for 12 h. After cell adhesion, each group was pretreated with a solution containing different drugs for 1 h before LPS (lipopolysaccharide) modeling. Cell modeling was then performed with 1 μg / mL LPS for 24 h, and the cell supernatant was collected. The levels of IL-1β, IL-6, and TNF-α inflammatory factors in the cell supernatant were detected using ELISA according to the kit instructions. Results are shown below. Figure 8 At a concentration of 10 μM, BA-6 exhibits anti-inflammatory effects in vitro, inhibiting the levels of inflammatory factors IL-1β, IL-6, and TNF-α in cell supernatant. The effect is significantly better than that of betulinic acid, suggesting that this compound has anti-inflammatory activity.

[0064] Example 9: Toxicity experiments of betulinic acid 28-O-β-D-glucoside (BA-6) and betulinic acid (BA) on THP-1 and RAW264.7 cells.

[0065] THP-1 cells were loaded at 6 × 10 4 THP-1 cells were seeded at a density of 2 × 10⁶ cells / well in 96-well plates. PMA (100 ng / mL) was added to induce differentiation of M0 macrophages into M1 macrophages for 12 h. After cell adhesion, each group was treated with solutions containing different drugs for 24 h. 10 μL of CCK-8 solution was added to each well, taking care to avoid air bubbles, and the plates were gently shaken and incubated in a cell culture incubator for 2 h. The absorbance (OD value) of the plates was then measured at 450 nm using a microplate reader, and the data were recorded to calculate the cell viability (%) at different drug concentrations. RAW264.7 cells were seeded at a density of 2 × 10⁶ cells / well.4 The cells were seeded at a density of 100 cells / well in a 96-well plate. After the cells naturally adhered to the plate, the subsequent procedures were the same as above.

[0066] At a concentration of 50 μM, betulinic acid 28-O-β-D-glucoside (BA-6) was found to be non-toxic at the cellular level (IC50 = 261.5 μM), while betulinic acid (BA) was cytotoxic. (See [link to relevant documentation]). Figure 9 .

[0067] Calculation formula: Cell viability = [(Cs-Cb) / (Cc-Cb)] × 100%;

[0068] Cs: Absorbance of experimental wells (including cell culture medium, CCK-8, and the compound to be tested);

[0069] Cc: Absorbance of control wells (including cell culture medium, CCK-8, and wells without the analyte compound);

[0070] Cb: Absorbance of blank wells (excluding cells and culture medium containing the test compound, CCK-8).

[0071] BA-6 is a glycoside derivative of BA, exhibiting significantly improved water solubility and bioavailability compared to BA. Furthermore, this derivative is easier to synthesize and has a higher yield. Our experimental results also suggest that BA-6 has protective and therapeutic effects on mice with DNCB-induced atopic dermatitis, with efficacy superior to dexamethasone. In addition, BA-6 effectively alleviates DNCB-induced atopic dermatitis in mice while also enhancing systemic immune function, demonstrating good safety. Therefore, the drug BA-6 provided by this invention is a potential treatment for atopic dermatitis.

[0072] This invention has been illustrated through specific embodiments, but these embodiments are only used to demonstrate preferred technical solutions and are not intended to limit the scope of protection of this invention. Those skilled in the art can, under the guidance of the core ideas of this invention and according to specific needs and technical background, make appropriate optimizations, adjustments, and improvements to the technical solutions of this invention, or make equivalent substitutions for some technologies. These adjustments, improvements, and equivalent substitutions all fall within the scope of protection of this invention.

Claims

1. The use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for treating skin inflammation, characterized in that: The skin inflammations mentioned include atopic dermatitis, contact dermatitis, eczema, and allergic dermatitis.

2. The application according to claim 1, characterized in that: The dosage of betulinic acid 28-O-β-D-glucoside is 0-10 mg / kg.

3. The application according to claim 2, characterized in that: The dosage of betulinic acid 28-O-β-D-glucoside is 0-5 mg / kg.

4. The application according to claim 1, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 1, characterized in that: The drug can be administered topically, orally, rectally, or as a parenteral drug.

6. A medicament or pharmaceutical composition for treating skin inflammation, characterized in that: The pharmaceutical composition contains betulinic acid 28-O-β-D-glucoside, and the skin inflammation includes atopic dermatitis, contact dermatitis, eczema, and allergic dermatitis.

7. The pharmaceutical composition according to claim 6, characterized in that: The dosage of betulinic acid 28-O-β-D-glucoside is 0-10 mg / kg.

8. The pharmaceutical composition according to claim 7, characterized in that: The dosage of betulinic acid 28-O-β-D-glucoside is 0-5 mg / kg.

9. The pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 6, characterized in that: The drug composition can be administered topically, orally, rectally, or as a parenteral drug.

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