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

Technical Field

[0001] The present invention belongs to the field of pharmacology of natural products, and particularly relates 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 Art

[0002] Inflammatory skin diseases are a group of skin inflammatory reactive diseases caused by various factors. Their pathogenesis is complex and may involve factors within the body, such as the skin microbiota, genetics, abnormal immune function, mental disorders, and daily lifestyle (including eating habits, smoking, drinking, and sleep). In addition, the skin may also trigger an inflammatory response after being stimulated by pathogens in the external environment. From the perspective of modern medicine, inflammatory skin diseases are mainly divided into 5 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, rosacea, etc. The clinical manifestations of these diseases vary, and their pathogenesis has not been fully clarified. However, it has been recognized that they share common characteristics: inflammation and immune factors are involved in the pathogenesis, including skin inflammatory responses mediated by T cells and antigen - antibody. The drug treatment methods for inflammatory skin diseases mainly include topical drugs and systemic treatment drugs. Topical drugs include glucocorticoids, calcineurin inhibitors, vitamin D3 derivatives, retinoids, antibiotics, etc.; systemic treatment drugs include antihistamines, glucocorticoids, traditional immunosuppressants, antibiotics, etc. In addition, phototherapy is also a commonly used treatment method. In recent years, with the in - depth study of the pathogenesis of inflammatory skin diseases, new treatment means such as biological agents and small - molecule targeted drugs have emerged continuously, bringing more choices and hopes to patients.

[0003] Atopic dermatitis (AD) is a recurrent, chronic, non-infectious inflammatory skin disease. AD typically presents as itchy, erythematous scaly plaques, crusting, fissuring, and lichenified lesions, often with seasonal fluctuations. The distribution of lesions differs between children and adult populations. It usually develops in childhood and may persist into adulthood. According to the Global Burden of Disease Study, the prevalence is 15% to 20% in children and as high as 10% in adults, making atopic dermatitis the 15th most common non-fatal disease and the skin disease with the highest disease burden, imposing a huge physical, psychological, and socioeconomic burden. 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 lymphoid cells, 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, mainly interleukin 4, interleukin 13, and interleukin 31, which activate the downstream Janus kinase (JAK) pathway. Cytokines promote inflammation, itching, and the production of antigen-specific IgE by activating B cells and plasma cells. Itching in atopic dermatitis is based on signal transduction between pruritogens released by keratinocytes, mast cells, and immune cells (T cells and eosinophils) and small sensory nerve fibers in the skin. Pruritogens include Th2 cytokines (especially interleukin 4, -13, and -31), thymic stromal lymphopoietin (an epithelial-derived pro-inflammatory cytokine), histamine, proteases, and neuropeptides. These pruritogens bind to receptors on sensory C and Aδ nerve fibers in the epidermis and dermis, which sense itching and pain.

[0004] Currently, the treatment methods for atopic dermatitis are selected according to the clinical stage of the disease (mild, moderate or severe), the extent of the body surface area involved, age, co-existing diseases and medications taken by the patient, the severity of itching, the degree of impaired quality of life, and the patient's goals. It mainly includes topical medications and oral medications. For mild atopic dermatitis, topical medications are mainly used, including glucocorticoids (TCS), calcineurin inhibitors (TCI), phosphodiesterase 4 inhibitors (PDE-4 inhibitors), Janus kinase inhibitors (JAK inhibitors); glucocorticoids (TCS) as the first-line treatment drug can quickly control the condition, but it should not be used over a large area for a long time. Long-term use may cause adverse reactions such as skin atrophy and barrier function damage, and even hormone-dependent dermatitis may occur; some patients may experience irritation reactions such as local burning pain, stinging or increased itching when using calcineurin inhibitors (TCI) and phosphodiesterase 4 inhibitors (PDE-4 inhibitors), and these symptoms may affect the patient's medication compliance. For moderate and severe atopic dermatitis, combination medications are required, including antihistamines, immunosuppressants such as cyclosporine, azathioprine and methotrexate, systemic glucocorticoids such as prednisone, and biological agents such as dupilumab and tralokinumab. Long-term use of glucocorticoids can cause metabolic disorders and lead to obesity, and some patients may develop hypertension, increasing the risk of cardiovascular diseases, etc.; the first-generation antihistamines such as diphenhydramine and chlorpheniramine have strong central inhibitory effects, and some antihistamines such as astemizole and terfenadine may cause cardiac toxicity; biological agents are usually expensive, and long-term use will bring a heavy economic burden to patients. Biological agents control inflammation by regulating the immune system, but at the same time may also increase the risk of infection, and some patients may have allergic reactions such as rashes, 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 at the same time may also increase the risk of infection, especially upper respiratory tract infections and herpes zoster, and JAK inhibitors may cause abnormal blood routine. Since the pathogenesis of AD has not been clearly defined, current drug treatments cannot cure the disease and there is a lack of specific drugs. Therefore, clinical treatment pays more attention to controlling the development of the pathological condition and improving the patient's quality of life. From the perspective of the comprehensive application effect, dexamethasone is mainly used clinically to treat AD. Summary of the Invention

[0005] In view of the problems existing in the existing drugs for treating dermatitis, such as limited efficacy, slow onset, many side effects, high long-term use risks, large individual differences, high treatment costs, and possible immune reactions, the present invention for the first time discloses the application of betulinic acid 28-O-β-D-glucoside (also known as betulinic acid 28-O-β-D-pyran glucoside, abbreviated as BA-6) in the prevention and treatment of dermatitis, with the technical progress of significantly improving the symptoms of dermatitis.

[0006] The present invention adopts the following technical solutions.

[0007] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for preventing and treating skin inflammation.

[0008] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for preventing and treating skin erythema and papules.

[0009] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for preventing and treating skin pruritus.

[0010] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for reducing skin edema and epidermal thickening.

[0011] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for reducing skin scales.

[0012] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for reducing skin lichenification.

[0013] Use of betulinic acid 28-O-β-D-glucoside in the preparation of a medicament for preventing and treating skin ulceration.

[0014] In the present invention, skin inflammation includes atopic dermatitis, contact dermatitis, eczema, paronychia, rash, psoriasis, lupus erythematosus, papulosquamous dermatosis, pityriasis rosea, seborrheic dermatitis, pemphigus, pemphigoid, acne, tinea corporis, impetigo, SAPHO syndrome, hidradenitis suppurativa, neurodermatitis, drug-induced dermatitis, lichen planus, pityriasis rubra pilaris, vitiligo, prurigo nodularis, systemic sclerosis, bedsore, granulomatous dermatosis, dermatomyositis, urticaria, chronic skin pruritus or alopecia areata; preferably, skin inflammation includes atopic dermatitis, contact dermatitis, eczema, rash, psoriasis, lupus erythematosus, papulosquamous dermatosis, pityriasis rosea, seborrheic dermatitis, vitiligo, acne and tinea corporis, etc. Betulinic acid 28-O-β-D-glucoside disclosed in the present invention can not only treat or prevent dermatitis and its caused symptoms, but also has the advantages of low cytotoxicity, safety and reliability.

[0015] The present invention discloses a pharmaceutical composition for treating skin inflammation, which takes betulinic acid 28-O-β-D-glucoside as an active ingredient; it may also include conventional pharmaceutical excipients, including one or several of diluents, dispersants, binders, lubricants, penetration enhancers.

[0016] Specifically, conventional pharmaceutical excipients refer to one or more compatible solid or liquid fillers or gel substances that can be used medicinally, have sufficient purity and low toxicity, and can be blended with each other and with the active ingredients of the present invention without reducing the efficacy of the active ingredients. Pharmaceutically acceptable carriers include, by way of example, cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, 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, glycerol, mannitol, sorbitol, etc.), cyclodextrins (such as hydroxypropyl-β-cyclodextrin), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0017] In the present invention, the dosage forms of the drug include pills, tablets, powders, pastes, ointments, granules, capsules, nano-formulations, aerosols, sprays, plasters, solutions, injections, sustained-release preparations, controlled-release preparations, gels or suppositories.

[0018] In the present invention, the drug or drug composition includes topical, oral, rectal or parenteral drugs. Preferably, the drug is a solution.

[0019] Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid compound present in various plant species, such as Pulsatilla chinensis in the Ranunculaceae family, Syzygium buxifolium in the Myrtaceae family, Nerium oleander, etc., and can also be prepared by selecting its metabolic precursor betulin. BA and its derivatives (synthesized by modifying at the C-3, C-20 and C-28 sites) have a variety of biological activities, including anti-cancer, anti-HIV, anti-parasitic and anti-angiogenic activities. In particular, it has been reported that BA has cytotoxic effects on several tumor cell lines of different origins as well as cancer animal models. Although BA has strong anti-HIV and anti-tumor activities, it is considered to have weak anti-inflammatory activity. According to relevant research reports, in lipopolysaccharide (LPS)-induced endotoxin shock, BA pretreatment exhibits anti-inflammatory activity through the mechanism of IL-10, but the anti-inflammatory activity and protective effect are weaker than those of dexamethasone; the BA derivative BA5 (chemical structural name: 3β-hydroxy-lup-20(29)-en-28-acyl-morpholine) protects mice from a lethal LPS attack and reduces edema in delayed-type hypersensitivity, but the effect is also weaker than that of dexamethasone. Betulinic acid 28-O-β-D-glucoside (BA-6) designed and synthesized in the present invention is a glycoside derivative of BA, and its water solubility and bioavailability are significantly improved compared with BA. In particular, BA-6 has an obvious effect of improving dermatitis, and the curative effect is significantly better than that of dexamethasone, which is unexpected to those skilled in the art. Description of the Drawings

[0020] Figure 1 It is a diagram of the method for establishing a mouse model of DNCB-induced dermatitis.

[0021] Figure 2 It is a statistical chart of the effect of BA-6 on the body weight (g) of 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.

[0022] Figure 3 It is a statistical chart of the effect of betulinic acid 28-O-β-D-glucoside on the dorsal skin of mice with DNCB-induced dermatitis and the EASI score. Among them, A is the dorsal skin of dermatitis mice, and B is the statistical chart of EASI score; 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.

[0023] Figure 4 It is a statistical chart of the effect of BA-6 on the ear skin of mice with DNCB-induced atopic dermatitis, as well as the difference in ear thickness and the difference in ear weight. Among them, A is the ear skin of dermatitis mice, B is the statistical chart of the difference in ear thickness of dermatitis mice, and C is the statistical chart of the difference in ear weight of dermatitis mice; 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.

[0024] Figure 5 It is a statistical chart of the effect of BA-6 on the spleen index of 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; comparison between the BA-6 group and the dexamethasone group (DEX, 3 mg / kg) group, &&& P<0.001; n≥7.

[0025] Figure 6Effects of BA-6 on inflammatory factors IFN-γ, IL-6, and IL-1β in the serum of DNCB-induced atopic dermatitis mice, where A is IFN-γ, B is IL-6, and C is 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 the BA-6 group with the dexamethasone group (DEX, 3 mg / kg), & P<0.05; n = 3.

[0026] Figure 7 Statistical chart of the survival rate of BA-6 on DNCB-induced atopic dermatitis mice.

[0027] Figure 8 ELISA experiment of BA-6 and betulinic acid (BA) on the release of inflammatory factors IL-1β, IL-6, and TNF-α induced by LPS, where A is IL-1β, B is IL-6, and C is TNF-α; compared with the normal control group, ### P<0.001, compared with the model group, *P<0.001, ***P<0.001; compared the BA-6 (10 μM) group with the betulinic acid (BA, 10 μM) group, $$ P<0.01, $$$ P<0.001; n = 3.

[0028] Figure 9 CCK-8 cytotoxicity experiment; a cell survival rate greater than 90% is usually considered non-toxic or low-toxic; A is the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) and betulinic acid (BA) at a concentration of 50 μM on the induction of THP-1 cells to differentiate into M1 macrophages; B is 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 is the cytotoxicity of betulinic acid 28-O-β-D-glucoside (BA-6) at concentrations of 50, 100, 150, 200, 250, and 300 μM on the induction of THP-1 cells to differentiate into M1 macrophages; D is the IC50 value of betulinic acid 28-O-β-D-glucopyranoside (BA-6), IC50 = 261.5 μM; n = 3. Detailed implementation method

[0029] Betulinic acid 28-O-β-D-glucoside (BA-6) disclosed by the present invention can not only treat or prevent dermatitis and its symptoms, but also has low cytotoxicity.

[0030] In order to make the objectives and technical solutions of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the data analysis is a conventional statistical analysis method; for those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions; the reagents and materials, unless otherwise specified, can all be obtained from commercial channels, and the animal experiments comply with the relevant requirements of Soochow University.

[0031] In the present invention, the chemical structures of betulinic acid 28-O-β-D-glucoside (Compound BA-6) and betulinic acid (Compound BA) are as follows:

[0032] Betulinic acid 28-O-β-D-glucoside can be obtained according to conventional methods. Examples of the present invention are as follows:

[0033] To a 25 mL acetic anhydride solution of 5.00 g D-glucopyranose, 125 mg of iodine was added, and the reaction was stirred magnetically at room temperature until the reaction system became brown and transparent. After the reaction was completed, 125 mL of dry dichloromethane was added to the reaction mixture for dilution, and 30 mL of an acetic acid solution of 33% hydrogen bromide was added under ice bath cooling. After addition, the reaction was stirred at room temperature until the reaction was completed as detected by TLC. The reaction mixture was diluted with 125 mL of dichloromethane and washed successively with ice water, saturated sodium bicarbonate solution, and saturated sodium thiosulfate solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a colorless oil, which was recrystallized from ether and petroleum ether to obtain 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 mixed solution of CH2Cl2 / H2O (76 ml / 76 ml). Successively, n-Bu4NBr (847 mg, 2.628 mmol) and K2CO3 (2.2 g, 16.421 mmol) were added thereto, and the reaction was stirred at room temperature for 6 h. Then, 100 ml of dichloromethane was added for dilution, 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 obtain 4.3 g of a white solid (G4) with a yield of 83%. G4 (4 g, 5.083 mmol) was dissolved in a mixed solution of THF / H2O (100 ml / 10 ml). Sodium hydroxide (1.2 g, 30.498 mmol) was added thereto, and the reaction was stirred at room temperature for 12 h. Then, 1M HCl was added to the reaction solution to adjust the pH to 4, and then water was added to precipitate the product, which was filtered and dried. It was redissolved in methanol and separated by a C18 chromatographic column (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%. The NMR and MS data are as follows: 11H 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). 13 13C NMR (101 MHz, (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 / z calcd for C 36 H 58 O8K [M + K] + 657.3763, found 657.3738. HPLC purity: 99.12%.

[0034] Example 1; Method for Establishing a Model of DNCB-Induced (Atopic) Dermatitis Mice BALB / c mice, SPF grade, female, 7 - 8 weeks old, weighing 18 - 21 g. The mice were adaptively fed for one week. After weighing, the mice were 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), BA - 6 (10 mg / kg). Prepare the modeling agents for the back and ears: Use acetone: olive oil = 4:1 as the solvent and DNCB as the solute. Prepare 5% DNCB modeling agent for the back and 1% DNCB modeling agent for the ears respectively. Prepare the drug solution: Weigh 60 mg of BA - 6 and prepare it with a mixed solvent of ethanol: water: glycerol = (70:28:2, volume ratio) at a concentration of 1 mg / mL. Other dosing doses are prepared proportionally. Two days before the experiment, use a hair clipper to shave the back hair of the mice. The shaving area is about 3 cm × 3 cm, and then use depilatory cream to remove the surface fluff on the back. On the 1st day, the sensitization stage: Except for the normal control group, at 2 pm, 50 μL of 5% DNCB solution was evenly applied to the back of each mouse in the other groups to sensitize the back skin of the mice. The normal control group was given the solvent. Repeat the operation on the 1st day on the 2nd day to resensitize the back skin of the mice. The normal control group was given the solvent. On the 3rd day, the challenge stage: Except for the normal group, at 2 pm, 100 μL of 1% DNCB solution was given to the right ear of each mouse in the other groups for challenge, with 50 μL on each of the inner and outer sides of the right ear of the mouse. Repeat the operation on the 3rd day on the 4th and 5th days to continuously challenge the right ear of the mice for three days to induce and establish a mouse model of atopic dermatitis. The manifestations of successful model establishment are that under the repeated stimulation of the DNCB solution, the back skin of the mice shows redness, ulceration, exudation, papules, desquamation and sclerosis; the ear skin shows redness, erosion, sclerosis, etc. Administration method: Mice in the dexamethasone (DEX, 3 mg / kg) group were given dexamethasone cream by applying it to the back and the inner and outer sides of the right ear of the mice at 4 pm every day (days 1 - 7). Mice in the BA - 6 (2.5 mg / kg), BA - 6 (5 mg / kg), and BA - 6 (10 mg / kg) groups were given the drug - containing solution by applying it to the back and the inner and outer sides of the right ear of the mice at 10 am and 4 pm every day (days 1 - 7).

[0035] The normal control group and the model group were given an equal amount of a mixed solvent of ethanol: water: glycerol = (70:28:2) by applying it to the back and the inner and outer sides of the right ear of each mouse at the same time as the above - mentioned dosing groups to keep the overall behavior consistent with the dosing groups. On the 8th day, after measuring the thickness of the left and right ears of each mouse, the mice were sacrificed, and the spleen and mouse ears were taken to calculate the spleen index and the difference in ear weight and ear thickness (use a 6 - mm - diameter punch to punch ear discs at the same part and weigh them); The experimental process is as Figure 1 shown.

[0036] Example 2: Effect of betulinic acid 28-O-β-D-glucoside on the body weight of DNCB-induced atopic dermatitis mice The body weight changes of each group of mice were statistically analyzed every day and plotted using GraphPad Prism 8.3.0. As Figure 2 shown, the body weight of the normal control group of mice was stable, while the body weight of the DNCB-induced model group of mice decreased sharply from the 3rd day of modeling. There was a significant difference between the 3rd - 6th days of modeling and the normal control group (P < 0.01), which was in line with the symptoms of DNCB-induced atopic dermatitis mice. Compared with the model group, there was a significant difference in the BA-6 (2.5 mg / kg) administration group on the 5th day (P < 0.05), and there was no significant difference in the dexamethasone group; compared with the dexamethasone group, there were significant differences in the BA-6 (2.5 mg / kg) administration group on the 5th and 8th days (P < 0.05), and there were significant differences in the BA-6 (5 mg / kg) and BA-6 (10 mg / kg) administration groups on the 8th day (P < 0.05). Therefore, BA-6 improved the trend of body weight loss in DNCB-induced atopic dermatitis mice, and its curative effect was better than that of dexamethasone.

[0037] Example 3: Effect of betulinic acid 28-O-β-D-glucoside on the lesion area size, skin lesion severity and comprehensive score (EASI) of the back skin of DNCB-induced atopic dermatitis mice When the local skin tissue comes into contact with DNCB, DNCB binds to the proteins on the surface of skin cells to form a hapten, activating the immune system, and ultimately leading to typical symptoms such as skin redness, itching, exudation, ulceration, and capillary dilation. The EASI score is specifically as follows: A scoring system consisting of 4 symptom indicators of erythema, papules / edema, scales, and lichenification / crusting is shown in Table 1. Each indicator is divided into 0 - 3 points according to its severity, where no symptoms are set as 0 points, mild symptoms are set as 1 point, moderate symptoms are set as 2 points, and severe symptoms are set as 3 points. After scoring each indicator, the sum of all scores is regarded as the total score, and the highest total score is 12 points.

[0038] As Figure 3As shown, during the experiment, the dorsal skin of mice in each group was observed and photographed on the 1st, 3rd, 5th, and 7th days. It was found that starting from the 2nd day 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 challenge, obvious swelling, crusting, hardening, and lichenification lesions appeared on the dorsal skin of mice in the model group, while the dorsal skin of mice in the normal group remained smooth and intact. Compared with the model group, the skin damage symptoms of mice in the BA-6 administration group were alleviated during the same period. There was no obvious swelling and exudation on the skin, no scales were seen, and erythema, papules, and skin hardening were significantly reduced. Signs of scab shedding from the damaged skin and the generation of new skin began to appear on the 5th day. The swelling symptoms of the dorsal skin of mice in the dexamethasone administration group were alleviated on the 3rd day, but similar ulceration, aggravated hardening, and increased swelling occurred in the later stage compared with the model group. Compared with the normal control group, the results of the EASI score of the dorsal skin of mice in the model group were statistically significant on the 3rd, 5th, and 7th days (P<0.001). Compared with the model group, the growth rate of the EASI score of the dorsal skin of mice in the BA-6 administration group was slow starting from the 3rd day, and all were statistically significant (P<0.001). The EASI score of the dorsal skin of mice in the dexamethasone group was lower than that of the model group on the 3rd day (P<0.05), but there was no difference from the model group on the subsequent 5th and 7th days. Compared with the dexamethasone group (DEX), the improvement degree of the dorsal skin of mice in the BA-6 administration group was better, and the EASI score was statistically significant on the 3rd, 5th, and 7th days (P<0.05). The above data indicate that BA-6 has a better therapeutic effect on the dorsal skin of AD mice than dexamethasone.

[0039] Table 1 EASI score

[0040] Example 4: Effects of BA-6 on the lesion area size, skin lesion severity, ear thickness, and ear weight difference of the ear skin of DNCB-induced atopic dermatitis mice On the 8th day, the left and right ears of mice in each group were photographed. The thickness of the left and right ears of mice in each group was measured with a vernier caliper (when measuring, follow the principle of reading at the same position on each ear to minimize errors). The calculation formula for the ear thickness difference: ear thickness difference = right ear thickness - left ear thickness. After sacrificing the mice on the 8th day, the ears of the mice were taken. The two ears of the mice were overlapped, and a punch with a diameter of 6 mm was used to punch ear round pieces at the same part. The left and right ear round pieces were weighed separately. The calculation formula for the ear weight difference: ear weight difference = right ear weight - left ear weight. As Figure 4As shown, the right ears of the mice in the normal group were smooth, with clear blood vessel choroids and no swelling, redness or scab formation. Compared with the normal control group, the right ears of the mice in the model group were swollen and scabbed. The differences in ear thickness and ear weight of the mice in the model group were relatively large, and there were statistical significances in the results of ear thickness and ear weight differences compared with those of the normal control group (P<0.001), indicating that the ears of the mice in the model group were severely edematous and thickened. Compared with the model group, the differences in ear thickness and ear weight of the mice in all drug administration groups decreased, with statistical significances (P<0.001). Among them, compared with the ear thickness of the dexamethasone group, BA-6 was more obvious in reducing the ear weight and decreasing the ear thickness difference of the mice, and there was a statistical significance (P<0.05). The above data indicate that BA-6 can significantly improve DNCB-induced ear swelling in mice, reduce the ear weight and ear thickness difference, and its curative effect is better than that of dexamethasone.

[0041] Example 5: Effect of BA-6 on spleen index of mice with atopic dermatitis induced by DNCB DNCB-induced skin inflammation mainly activates Th2 cells and may also activate the systemic immune system, leading to the proliferation and activation of immune cells in the spleen and resulting in an increase in spleen volume. As Figure 5 shown, compared with the normal group, the spleen index of the mice in the model group was significantly increased, with statistical significance (P<0.001). Compared with the model group, the spleen index of the BA-6 administration group decreased. Among them, there was a statistical significance in the spleen index of the BA-6 (5 mg / kg) administration group compared with that of the model group (P<0.01). The value of the spleen index of the dexamethasone group was extremely low, even significantly lower than that of the normal control group and there was a statistical significance (P<0.001). Compared with the spleen index of the dexamethasone group, the spleen index of the BA-6 administration group also had a statistical significance (P<0.001). This indicates that dexamethasone produced immunosuppression in mice, while BA-6 had an immunomodulatory effect on mice without immunosuppressive effect, suggesting that the drug had a positive regulatory effect on the pathological state or immune function of the spleen and improved the immunity of mice when stimulated by stress, inflammation, etc.

[0042] Example 6: Effect of BA-6 on inflammatory factors in serum of mice with dermatitis On the 8th day, all the mice were sacrificed by cervical dislocation after blood collection from the eyeballs. The whole blood was allowed to sediment naturally at 4°C for 2 h, centrifuged at 4000 rpm and 4°C for 15 min to collect the upper serum, and the expressions of IFN-γ, IL-6 and IL-1β inflammatory factors in the serum were detected by ELISA according to the operation procedure of the kit instructions. As Figure 6As shown, compared with the normal control group, the levels of IFN-γ, IL-6, and IL-1β in the serum of DNCB-induced atopic dermatitis mice were significantly increased; compared with the model group, the inflammatory factors in the serum of the drug-administered groups of mice were decreased; among them, compared with dexamethasone, the BA-6 (5 mg / kg) drug-administered group had a more obvious decrease in IL-6, with a significant difference (P<0.05). The above data indicate that BA-6 improves DNCB-induced atopic dermatitis mice by reducing the inflammatory factors in the serum.

[0043] Example 7: Effects of BA-6 on the survival rate and behavior of DNCB-induced atopic dermatitis mice The number of surviving BABL / c mice in each group was statistically analyzed every day and plotted using graph prism 8.3.0. As Figure 7 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 showed that the dexamethasone group had an excessive immunosuppressive effect that exacerbated the death of atopic dermatitis mice, while BA-6 had a protective effect and inhibited the death of DNCB-induced acute atopic dermatitis mice. The scratching behavior of the model group mice increased significantly, while the scratching behavior of the BA-6 group and dexamethasone group mice decreased significantly, especially the scratching behavior of the BA-6 groups at each dose was less than that of the dexamethasone group.

[0044] Example 8: In vitro anti-inflammatory experiments of BA-6 and betulinic acid THP-1 cells were seeded in 24-well plates at a density of 5×10 5 cells / well, and 100 ng / mL phorbol-12-Myristate-13-Acetate (PMA) was added to induce THP-1 cells for 12 h. After the cells adhered, before LPS (lipopolysaccharide) modeling, the cells were pretreated with solutions containing different drugs for 1 h, and 1 μg / mL LPS was used for modeling for 24 h. The cell supernatant was taken. The contents of IL-1β, IL-6, and TNF-α inflammatory factors in the cell supernatant were detected by ELISA according to the operation procedure of the kit instructions. The results are shown in Figure 8 , at a concentration of 10 μM, BA-6 had an anti-inflammatory effect in vitro, inhibiting the contents of IL-1β, IL-6, and TNF-α inflammatory factors in the cell supernatant, and the effect was significantly better than that of betulinic acid, suggesting that this compound has anti-inflammatory activity.

[0045] Example 9: Toxicity Experiments of Betulinic Acid 28-O-β-D-Glucoside (BA-6) and Betulinic Acid (BA) on THP-1 Cells and RAW264.7 Cells Seed THP-1 cells in a 96-well plate at a density of 6×10 4 cells / well, add 100 ng / mL phorbol 12-myristate 13-acetate (PMA) to induce THP-1 cells for 12 h to induce the cells to differentiate from the M0 type to the M1 type of macrophages. After the cells adhere to the wall, add solutions containing different drugs to each group to treat the cells for 24 h. Add 10 μL of CCK-8 solution to each well, be careful not to generate bubbles, gently shake well, and incubate in a cell culture incubator for 2 h. Then use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) of this plate at a wavelength of 450 nm and record the data, and calculate the cell survival rate (%) at different drug concentrations. Seed RAW264.7 cells in a 96-well plate at a density of 2×10 4 cells / well. After the cells adhere to the wall naturally, the subsequent operations are the same as above.

[0046] It was found that at a concentration of 50 μM, betulinic acid 28-O-β-D-glucoside (BA-6) is non-toxic at the cellular level, IC 50 =261.5 μM, while betulinic acid (BA) is cytotoxic, see Figure 9 .

[0047] Calculation formula: Cell survival rate = [(Cs - Cb) / (Cc - Cb)]×100%; Cs: Absorbance of the experimental well (including cell culture medium, CCK-8, and the compound to be tested); Cc: Absorbance of the control well (including cell culture medium, CCK-8, without the compound to be tested); Cb: Absorbance of the blank well (cell-free and compound-free culture medium, CCK-8).

[0048] BA-6 is a glycoside derivative of BA, and its water solubility and bioavailability are significantly improved compared with BA. In addition, the synthesis of this derivative is easy and the yield is high. The results of this experiment also suggest that BA-6 has a protective and therapeutic effect on DNCB-induced atopic dermatitis mice, and its curative effect is better than that of dexamethasone. In addition, while effectively relieving DNCB-induced atopic dermatitis in mice, BA-6 can also improve the systemic immune function and show good safety. Therefore, the drug BA-6 provided by the present invention is a drug with the potential to treat atopic dermatitis.

[0049] The present invention has been illustrated by specific embodiments, but these embodiments are only used to demonstrate the preferred technical solutions of the present invention and do not limit the protection scope of the present invention. Those skilled in the art can, under the guidance of the core idea of the present invention, make appropriate optimization, adjustment and improvement to the technical solutions of the present invention according to specific requirements and technical backgrounds, or make equivalent substitutions for some technologies. These adjustments, improvements and equivalent substitutions all fall within the protection scope of the present invention.

Claims

1. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for preventing and treating skin inflammation.

2. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for preventing and treating skin erythema and papules.

3. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for preventing and treating skin pruritus.

4. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for reducing skin lesions and skin ulceration.

5. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for reducing skin scales and skin thickening.

6. Use of betulinic acid 28-O-β-D-glucoside in the preparation of a drug for reducing skin lichenification.

7. The application according to any one of claims 1 to 6, characterized in that, The dosage forms of the drug include pills, tablets, powders, pastes, ointments, granules, capsules, nano-formulations, aerosols, sprays, plasters, solutions, injections, sustained-release preparations, controlled-release preparations, gels or suppositories.

8. The application according to any one of claims 1 to 7, characterized in that, The drug includes topical, oral, rectal or parenteral drugs.

9. The application according to claim 1, wherein Skin inflammation includes atopic dermatitis, contact dermatitis, eczema, paronychia, rash, psoriasis, lupus erythematosus, papulosquamous dermatosis, pityriasis rosea, seborrheic dermatitis, pemphigus, pemphigoid, acne, tinea corporis, impetigo, SAPHO syndrome, hidradenitis suppurativa, neurodermatitis, drug-induced dermatitis, lichen planus, pityriasis rubra pilaris, vitiligo, prurigo nodularis, systemic sclerosis, bedsore, granulomatous dermatosis, dermatomyositis, urticaria, chronic skin pruritus or alopecia areata.

10. A pharmaceutical composition for treating skin inflammation, characterized in that, Using betulinic acid 28-O-β-D-glucoside as the active ingredient.

Images