A spirocyclic styracil compound, its preparation method and application

Abstract

This invention relates to the field of natural product pharmaceuticals, and more particularly to a spirobroussonin compound, its preparation method, and its application. The spirobroussonin C provided by this invention is isolated from the paper mulberry tree. This compound can inhibit the release of the inflammatory factor IL-13 from human keratinocytes induced by IL-4 / TNF-α, exhibiting activity superior to the clinical drug dexamethasone and without cytotoxicity. It can significantly alleviate ear dryness and excessive angiogenesis in MC903-induced atopic dermatitis mice, reduce ear swelling, epidermal thickening, and transepidermal water loss, alleviate the trend of weight loss, reduce mast cell infiltration, and inhibit the expression of serum IgE and Th2-type cytokines such as IL-13, IL-4, and TSLP in ear tissue. No glucocorticoid-like side effects such as skin atrophy or metabolic disorders were observed with Spirobroussonin C within the experimental dose range.

Description

Technical Field

[0001] This invention relates to the field of natural product pharmaceutical technology, and in particular to a spirocyclic phytosanitary compound, its preparation method, and its application. Background Technology

[0002] Atopic dermatitis (AD) is a chronic, relapsing inflammatory disease characterized by recurrent episodes of persistent itching and eczematous dermatitis. As the leading cause of dermatological disease, AD affects patients throughout their lives and carries a risk of comorbidities such as asthma and allergic rhinitis. Patients suffer from long-term, recurrent, severe itching, skin lesions, sleep disturbances, and social, psychological, and emotional dysfunctions. The pathogenesis of AD is highly complex, with studies indicating that type 2 inflammatory responses are involved in the entire process. Whether the disease is caused by impaired skin barrier function, abnormal immune responses, or the interaction of two factors, activation of type 2 inflammatory pathways and their driving factors IL-4 and IL-13 play a crucial role. Chronic itching is the most prominent symptom of AD. Its pruritogens include Th2 cytokines (especially IL-4, IL-13, and IL-31), TSLP, IL-33, histamine, proteases, and neuropeptides. These bind to some sensory nerves in the skin, leading to neurosensitization and generating "itching" signals in the brain. Therefore, "causal treatment" targeting type 2 inflammation is particularly crucial for the treatment and control of AD.

[0003] Traditional treatments for Alzheimer's disease (AD) have included topical corticosteroids (TCS), topical calcineurin inhibitors, oral antihistamines, immunosuppressants, and systemic hormones. TCS remains the first-line treatment, but long-term, large-area use can lead to skin thinning, atrophy, impaired barrier function, and even secondary infections, potentially resulting in steroid-dependent dermatitis. Relapse is also highly likely after discontinuation. In children with AD, TCS also presents challenges in adherence. In recent years, biologics and various small-molecule drugs have been approved, including IL-4Rα inhibitors, IL-13 inhibitors, and Janus kinase inhibitors, ushering in an era of precision medicine for AD. However, biologics have limitations such as high cost, the need for injection, and poor treatment adherence; while existing targeted therapies have clearly defined targets, they are costly, have significant side effects, and their long-term efficacy remains to be observed.

[0004] In recent years, experts both domestically and internationally have continuously emphasized and promoted the concept of comprehensive management of Alzheimer's disease (AD). How to prevent and reduce AD ​​recurrence and improve patients' quality of life has become a key issue that urgently needs to be addressed in clinical practice. Traditional Chinese medicine (TCM) offers advantages in the prevention and treatment of AD due to its multi-target nature, safety, barrier repair capabilities, and cost-effectiveness. It significantly ensures long-term efficacy and reduces recurrence rates. Extracting safe and stable effective components or discovering novel active molecules from Chinese herbal medicines can provide new options for combination or maintenance therapy of AD.

[0005] Paper mulberry (Broussonetia papyrifera) is a plant belonging to the genus Broussonetia in the family Moraceae, and it is abundant in my country. The Compendium of Materia Medica and the Records of Famous Physicians record that its stems and branches have effects such as dispelling wind and relieving itching, treating urticaria, and treating hives. The Wa ethnic medicine of China records that young branches of paper mulberry can treat psoriasis and neurodermatitis. Current research on this plant indicates that its chemical components involve flavonoids, lignans, phenylpropane, and terpenoids, mainly flavonoids. Pharmacological studies primarily focus on its anticancer, antioxidant, and anti-inflammatory activities. Studies have shown that extracts from paper mulberry roots have significant anti-atopic dermatitis activity.

[0006] To date, there are no reports on spirobroussonin C, a spirocyclic spirobroussonin compound, nor on its activity and applications. Summary of the Invention

[0007] The purpose of this invention is to provide a novel compound, spirobroussonin C, its preparation method, and its applications. This invention utilizes natural product chemical separation techniques to isolate and identify a novel spirobroussonin-like compound from the stems and branches of *Broussonetia papyrifera*. Furthermore, this invention conducted an IL-13 secretion inhibition experiment on IL-4 and TNF-α-stimulated HaCaT cells, showing that spirobroussonin C's activity was significantly superior to the clinically commonly used glucocorticoid dexamethasone. In addition, this invention evaluated its in vitro efficacy using an MC903-induced atopic dermatitis (AD) mouse model, showing that spirobroussonin C significantly reduced ear swelling, alleviated ear dryness and excessive angiogenesis in mice, mitigated the weight loss trend in AD mice, significantly reduced transepidermal water loss (TEWL) values, markedly reduced epidermal thickness in lesions, reduced mast cell infiltration, and inhibited serum IgE and ear tissue inflammatory factor levels; it can be used to prepare drugs for the prevention and / or treatment of inflammation, or drugs for the prevention and / or treatment of atopic dermatitis.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a spirobroussonin C compound, the compound being spirobroussonin C, with the following structural formula:

[0009] .

[0010] This invention also provides a method for preparing the spirocyclic mulberry alkaloid compound, the method comprising the following steps: taking the dried aerial parts of the mulberry tree, crushing them, extracting them with an ethanol solution, recovering the solvent to obtain an extract, dispersing the extract in water, and sequentially extracting it with petroleum ether or n-hexane, chloroform or dichloromethane or diethyl ether, ethyl acetate and n-butanol, wherein the chloroform or dichloromethane or diethyl ether extract is concentrated under reduced pressure and then loaded onto a normal-phase silica gel column, and eluted with a gradient using a nonpolar-weakly polar binary mixed mobile phase to obtain five fractions Fr. A-Fr. E;

[0011] The Fr. C fraction was separated by reversed-phase chromatography with gradient elution using a polar mobile phase, followed by gel column chromatography and then purification by semi-preparative HPLC with elution using a polar mobile phase to obtain the spirocyclic morpholine compound.

[0012] Preferably, the concentration (v / v) of the ethanol, methanol, or acetone solution is 50%-100%; the amount used is 5-8 times the mass of the paper mulberry powder, and the extraction is performed 1-4 times.

[0013] Preferably, in the nonpolar-weakly polar binary mixed mobile phase, the nonpolar solvent is selected from one or more of petroleum ether, n-hexane, and cyclohexane; the weakly polar solvent is selected from one or more of ethyl acetate, diethyl ether, chloroform, dichloromethane, acetone, and isopropanol, and the gradient range between the nonpolar solvent and the weakly polar solvent is 1:0 → 0:1.

[0014] Preferably, the Fr. C fraction is separated using an MCI reversed-phase column with gradient elution of methanol / water or acetonitrile / water at a ratio of 0:1 to 1:0, and the fractions are combined to obtain six fractions: Fr. C-1 to Fr. C-6.

[0015] Preferably, the Fr. C-1 fraction was separated by Sephadex LH-20 gel column chromatography with methanol elution, and then purified by semi-preparative HPLC using an RP-C18 column with methanol / water or acetonitrile / water at a volume ratio of 20-40:80-60 as the mobile phase to obtain the spirocyclic morpholinoid compound.

[0016] Furthermore, the present invention provides a pharmaceutical composition comprising the compound and / or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. When the compound of the present invention is used to prepare a drug, it can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99.0%, preferably 0.1-1%, of the compound spirobroussonin C and / or a pharmaceutically acceptable salt thereof, with the remainder being a pharmaceutically acceptable, non-toxic, inert, and pharmaceutically acceptable carrier and / or excipient. The pharmaceutical carrier or excipient is one or more solid, semi-solid, and liquid diluents, fillers, and pharmaceutical excipients.

[0017] The compounds or pharmaceutical compositions provided by the present invention are used in the form of a dose per unit body weight; the drugs of the present invention can be administered in various forms (liquid preparations, solid preparations, topical preparations, sprays, compound preparations, etc.); preferably administered in the form of topical preparations.

[0018] Furthermore, the topical formulation comprises the compound spirobroussonin C and / or its pharmaceutically acceptable salts and pharmaceutical carriers and / or excipients, wherein the content of spirobroussonin C and / or its pharmaceutically acceptable salts in the topical formulation is 0.1-1% w / w.

[0019] Furthermore, the topical preparation is an ointment, cream, gel, powder, liquid, or lotion.

[0020] Preferably, the composition can also be combined with other active pharmaceutical ingredients to exert a synergistic anti-inflammatory effect through multi-target regulation (such as inhibiting the COX-2 / NF-κB pathway, targeting inflammatory cytokines such as IL-4 / IL-13, IL-31, IL-33, IL-17, TNF-α, TSLP, etc., and inhibiting serum IgE, etc.), significantly enhancing the efficacy of single drugs.

[0021] The present invention also provides the use of the said compound or the said pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of inflammation.

[0022] The present invention also provides the use of the compound or the pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of atopic dermatitis.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. This invention has isolated and identified a new spirobroussonin C compound from the stems and branches of Broussonetia papyrifera. The raw material is readily available, and the separation and purification method is simple and suitable for industrial operation.

[0025] 2. The novel compound spirobroussonin C provided by this invention, when used in HaCaT cells stimulated by IL-4 and TNF-α, showed an IL-13 content of 16.18 ± 0.2 pg / mL, which was lower than that of the positive control drug dexamethasone (17.31 ± 0.6 pg / mL). This indicates that the compound has a significant anti-inflammatory effect, which is superior to that of the positive control drug dexamethasone.

[0026] 3. The novel compound spirobroussonin C provided by this invention can significantly reduce ear swelling induced by MC903 in mice with atopic dermatitis, alleviate ear dryness and excessive angiogenesis, and mitigate the weight loss trend in mice with atopic dermatitis. It also significantly reduces transepidermal water loss (TEWL), decreases epidermal thickness, reduces mast cell infiltration, and inhibits the expression of serum IgE and Th2-type cytokines such as IL-13, IL-4, and TSLP in ear tissue. No glucocorticoid-like side effects such as skin atrophy and metabolic disorders were observed with Spirobroussonin C within the experimental dosage range. This indicates that the compound has outstanding anti-atopic dermatitis activity. Attached Figure Description

[0027] Figure 1 The chemical structural formula of spirobroussonin C is shown below.

[0028] Figure 2 For the compound spirobroussonin C 1 H NMR spectrum;

[0029] Figure 3 For the compound spirobroussonin C 13 C NMR spectrum;

[0030] Figure 4 The HR-ESI-MS spectrum of the compound spirobroussonin C;

[0031] Figure 5 Images of skin lesions in the ears of mice;

[0032] Figure 6 SCORAD scores for different doses of spirobroussonin C treatment groups;

[0033] Figure 7 Different doses of spirobroussonin C treatment improved ear swelling in mice;

[0034] Figure 8 The effect of different doses of spirobroussonin C treatment on the TEWL value of mouse ear skin;

[0035] Figure 9 To improve skin epidermal thickness and mast cell infiltration in different spirobroussonin C treatment groups, A shows the HE staining results of each group, and B shows the toluidine blue staining results of each group.

[0036] Figure 10 The effects of different doses of spirobroussonin C treatment on the biomarkers IL-4 (A), IL-13 (B), IL-33 (C), TSLP (D), IL-6 (E), H and IgE (F) in serum and ear tissue were investigated. Detailed Implementation

[0037] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these solutions are provided to make the understanding of the disclosure of the present invention more thorough and complete.

[0038] Unless otherwise stated, the technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and does not limit the scope of the invention in any way. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0039] Example 1: Preparation method of compound spirobroussonin C

[0040] Take the dried aerial parts of the paper mulberry tree, crush them, and extract them three times with 6 times the amount of 90% ethanol, each time for 3 days. Combine the extracts and recover the solvent under reduced pressure to obtain the total extract.

[0041] The total extract was dispersed in water and extracted sequentially with petroleum ether or n-hexane or cyclohexane, chloroform or dichloromethane or diethyl ether, ethyl acetate, and n-butanol. The chloroform or dichloromethane or diethyl ether fraction was concentrated under reduced pressure and then loaded onto a normal-phase silica gel column. The mobile phase consisted of petroleum ether / ethyl acetate at volume ratios of 80:1, 50:1, 20:1, 10:1, 5:1, 1:1, 1:2, and 0:1, with gradient elution to obtain five fractions Fr. A→Fr. E. The mobile phase of petroleum ether / ethyl acetate could be replaced with n-hexane / ethyl acetate, n-hexane / acetone, cyclohexane / ethyl acetate, cyclohexane / acetone, petroleum ether / acetone, etc.

[0042] The Fr. C fraction was separated using an MCI reversed-phase column (small-pore resin gel column, polystyrene-based reversed-phase resin packing material) with gradient elution using methanol / water as eluent (volume ratios of 70:30, 80:20, 90:10, 100:0, 3-5 column volumes for each gradient). The fractions were then combined under thin-layer chromatography (developing solvent: petroleum ether / ethyl acetate, volume ratio 8:2) to obtain six fractions: Fr. C-1 → Fr. C-6. Acetonitrile / water can be used instead of methanol / water.

[0043] Fr. C-1 was separated by Sephadex LH-20 gel column chromatography (hydroxypropyl dextran gel column) (methanol elution), followed by semi-preparative HPLC purification using an RP-C18 column and a mobile phase of methanol / water (or acetonitrile / water) at a volume ratio of 25-40:75-60 to obtain the compound spirobroussonin C. Figure 1 As shown.

[0044] Example 2 Structural characterization of compound spirobroussonin C

[0045] The physical parameters and spectral data of compound spirobroussonin C are as follows: pale red amorphous powder, molecular formula C 16 H 14 O4, ,UV(MeOH)λ max (log ε )203(4.43),223(3.98),287(3.84)nm,IR(KBr)ν max 3434cm -1 1653cm -1 1586cm -1 1232cm -1 1128 cm -1 HR-ESI-MS ( m / z ),271.0962 [M+H] + (calcd. for C 16 H 15 O4, 271.0965), 1 H NMR and 13 The C10 NMR data for the compound spirobroussonin are shown in Table 1. 1 The H NMR spectrum is shown in [reference]. Figure 2 , compound spirobroussonin C 13 The C NMR spectrum is shown below. Figure 3The HR-ESI-MS spectrum of compound spirobroussonin C is shown in [reference needed]. Figure 4 .

[0046] Table 1. Compound spirobroussonin C 1 H and 13 C NMR data

[0047]

[0048] Note: a Data were determined using CD3OD as the solvent. 1 H NMR 600 MHz, 13 C NMR 150 MHz).

[0049] The final chemical structural formula of spirobroussonin C is as follows:

[0050] .

[0051] Example 3: Cellular Experiments on Anti-inflammatory Activity

[0052] In this embodiment, human immortalized keratinocytes (HaCaT) were purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured in Dulbecco modified DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. IL-4 and TNF-α were purchased from Wuhan Sanying Biotechnology Co., Ltd., China; CCK-8 reagent was purchased from MedChemExpress, USA; streptomycin and penicillin solutions were purchased from Saive Biotechnology Co., Ltd., China; dexamethasone was purchased from Shanghai Taoshu Biotechnology Co., Ltd., China; and the human IL-13 ELISA kit was purchased from Wuhan Yilairuit Biotechnology Co., Ltd., China.

[0053] This application yielded several compounds extracted from the paper mulberry tree. This embodiment lists the anti-inflammatory activity detection process for three of these compounds. Besides spirobroussonin C, the other two compounds are spirobroussonin A and taxifolin, with the following structural formulas:

[0054]

[0055] HaCaT cell viability assay: An atopic dermatitis HaCaT cell model was constructed using combined IL-4 and TNF-α induction. HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-drug antibiotics in a 37°C, 5% CO2 incubator. HaCaT cells in the logarithmic growth phase were then cultured at a rate of 1.2 × 10⁻⁶ cells / cells. 4 Cells were seeded per well in 96-well plates and incubated for 24 hours to allow for cell adhesion and normal growth. Control, model, positive control, and compound groups (spirobroussonin C, spirobroussonin A, or dihydroquercetin) were established. The control group received complete culture medium; the model group received medium containing 50 ng / mL IL-4 and TNF-α; the positive control group received 25 μg / mL dexamethasone; and the compound groups consisted of three groups, receiving 25 μg / mL spirobroussonin C, 25 μg / mL spirobroussonin A, and 25 μg / mL dihydroquercetin, respectively. Each concentration was used in six replicates, and the cells were cultured for another 24 hours. Then, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated again for 1 hour. After incubation, the absorbance (A) of each well was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula.

[0056] Cell viability = [(A s -A b ) / (A c -A b )]×100%;

[0057] In the formula: A s Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); A c Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but without drugs); A b The absorbance is for blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0058] IL-13 secretion inhibition assay: Cells in logarithmic growth phase were cultured at 8 × 10⁸ cells per well. 3 Cells were seeded at a density of [number] cells per well and incubated in 96-well plates for 24 h. Except for the control group, all cells were incubated with IL-4 and TNF-α for 24 h as described above to establish a cell model. Then, dexamethasone and the corresponding compound were added separately for positive control, and incubated for 24 h. The IL-13 content in the cell supernatant was then measured using a human IL-13 ELISA kit. Each concentration was tested in triplicate.

[0059] Experimental results showed that the compound spirobroussonin C had no inhibitory effect on HaCaT cell proliferation, indicating no cytotoxicity. Compared with the control group, the IL-13 content in the model group was significantly increased ( P < 0.001 indicates that the HaCaT cell model of atopic dermatitis was successfully established. Compared with the model group, the IL-13 content in the spirobroussonin C group was significantly reduced ( P The value < 0.001 indicates that spirobroussonin C significantly inhibits IL-13 secretion, demonstrating significant anti-inflammatory activity. The IL-13 content after treatment with Spirobroussonin C was 16.18±0.18 pg / mL, showing a superior inhibitory effect on IL-13 secretion compared to dexamethasone (17.95±0.10 pg / mL). In contrast, the IL-13 secretion levels of spirobroussonin A and taxifolin, also isolated from Broussonetia papyrifera, were slightly higher than those in the model group, indicating that neither compound inhibited IL-3. Specific results are shown in Table 2.

[0060] Table 2. Effects of different compounds on HaCaT cell survival and IL-13 secretion.

[0061]

[0062] Note: Compared with the control group, ### P < 0.001. Compared with the model group, *** P < 0.001.

[0063] Example 4 Animal Experiment

[0064] In this embodiment, 1 mg of MC903 dry powder was placed in 1 mL of anhydrous ethanol to prepare a 1 mg / mL stock solution, which was stored at -20°C. It was then diluted with anhydrous ethanol to prepare a 2 nmol MC903 modeling solution.

[0065] 1 mg of spirobroussonin C was placed in 1 mL of anhydrous ethanol to prepare a 1 mg / mL stock solution, which was stored at -20°C. Three high, medium, and low dose groups (150 μM, 100 μM, and 50 μM) were then prepared using anhydrous ethanol.

[0066] In this embodiment, 36 SPF-grade male BALB / c mice, 6 weeks old and weighing 20±2g, were provided by the Experimental Animal Center of Kunming Medical University and housed in the SPF-grade animal facility of the Experimental Animal Center of Kunming Medical University [Experimental Animal Use License No.: [SCXK (Yunnan) K2020-0004], in an environment with 12-hour light-dark cycle, constant temperature of 20-24 ℃ and relative humidity of 50%–60%, with free access to food and water.

[0067] In this embodiment, MC903 (calcipotriol analogue) was purchased from MedChemExpress, dexamethasone was purchased from Shanghai Taoshu Biotechnology Co., Ltd., mouse IgE, IL-4, IL-6, IL-13, IL-33 and TSLP ELISA kits were purchased from Wuhan Genemei Technology Co., Ltd., RIPA lysis buffer was purchased from Servicebio, and BCA protein quantification kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0068] Methods of grouping, modeling, and administering drugs to experimental animals

[0069] Mice were acclimatized for 7 days until they weighed approximately 23 g. They were then randomly divided into a normal control group (NC), an AD model group (MC), a high-dose spirobroussonin C group (SBC-H), a medium-dose group (SBC-Z), a low-dose group (SBC-D), and a positive control group (dexamethasone, DXM). Starting from day 1 of the experiment, 20 μL of anhydrous ethanol was applied to both ears of mice in the normal control group every morning using a pipette, while 20 μL of MC903 modeling solution was applied to both ears of mice in the other groups. This process was repeated for 10 consecutive days to induce the AD model.

[0070] Administration method: From day 4 to day 10, 4 hours after MC903 was applied to establish the model, 20 μL of the corresponding dose of spirobroussonin C solution was applied to both ears of mice in different dose spirobroussonin C treatment groups, compound dexamethasone cream was applied to the positive control group, and anhydrous ethanol was applied to the normal control group.

[0071] Routine indicators and skin lesion severity scoring: On days 0, 2, 4, 6, 8 and 10 after induction, the swelling of the left and right ears of mice in each group was measured at the same location using vernier calipers before modeling. The transcutaneous water loss (TEWL) value of each group of mice was measured using a transcutaneous water loss meter before modeling.

[0072] On day 11 of the experiment, SCORAD clinical scores were calculated based on the severity of ear skin symptoms. The clinical scores were based on the severity of skin lesions: 1) erythema / bleeding; 2) crusting / dryness; 3) edema / exudation; 4) abrasion / erosion. Each symptom severity was scored as 0 (none), 1 (mild), 2 (moderate), and 3 (severe). The severity of each symptom was assessed against reference photographs, and the sum of the scores (minimum 0 and maximum 12) was considered the dermatitis score.

[0073] Sample collection: On day 11 of the experiment, mice were anesthetized with isoflurane inhalation. Whole blood samples were collected via abdominal aortic puncture and allowed to stand at room temperature for 30 minutes to allow clotting. The samples were then centrifuged at 4 ℃ and 3000 ×g for 15 minutes, and the supernatant serum was collected and frozen at -80 ℃. Mice were euthanized by cervical dislocation, and ear skin tissue was separated, fixed in 10% neutral formalin solution, and subjected to histopathological analysis.

[0074] HE staining and toluidine blue staining: Ear tissue was fixed in 4% paraformaldehyde for 48 h, then rinsed with water overnight. The samples were then dehydrated, paraffin-embedded, and embedded to prepare sections for HE and toluidine blue staining. For HE staining, sections were stained with hematoxylin for 1 min, then eosin for 50 s, and then washed with a series of ethanol solutions. For toluidine blue staining, sections were stained with toluidine blue solution for 3–5 min and washed with distilled water. Each section was dehydrated with ethanol of different concentrations (from 70% to 100%) and soaked in xylene, and finally mounted with neutral resin. The epidermal thickness and mast cell count of each group of mice were observed using an optical microscope.

[0075] ELISA assay: Serum samples from mice in each group were analyzed according to the instructions of the commercially available ELISA kits to measure serum IgE levels. Ear tissue samples were ground into powder using liquid nitrogen. Subsequently, each sample was lysed at a 4:1 ratio with lysis buffer (containing 1% phosphatase inhibitor and 1% protease inhibitor). The samples were lysed three times on ice using a high-intensity sonicator. The resulting supernatant was centrifuged at 12000×g for 10 min at 4°C and then transferred to new centrifuge tubes. Protein concentration was then determined using a BCA kit. Cytokine levels were measured according to the instructions of the ELISA kits for inflammatory factors such as TSLP, IL-13, and IL-4. Cytokine levels are expressed relative to total protein concentration.

[0076] Statistical methods: All data were independently repeated at least three times and are expressed as mean ± standard deviation. Differences between groups were statistically assessed using one-way ANOVA combined with the Bonferroni post-hoc test. All results were visualized using GraphPad Prism 9.5.1 software.P A difference of < 0.05 is considered statistically significant.

[0077] Experimental results

[0078] Effects of compound spirobroussonin C on MC903-induced AD-like skin lesions in mice

[0079] Compared with the normal control group, mice in the MC903 treatment group showed obvious skin symptoms such as erythema, edema, crusting, scaling, and dryness. After one week of treatment with the compound spirobroussonin C, the MC903-induced ear dryness and excessive angiogenesis were significantly reduced. Figure 5 The SCORAD clinical score in the spirobroussonin C treatment group was statistically different from that in the model group. Figure 6 , P < 0.01). This indicates that spirobroussonin C can effectively prevent and alleviate atopic dermatitis-like skin lesions.

[0080] On the 4th day after MC903 treatment for modeling, ear swelling began to appear, and the ear thickness gradually increased over time. After treatment with the compound spirobroussonin C, the increase in ear thickness was slower than that in the model group, and the degree of ear swelling was significantly reduced after the modeling was completed. Figure 7 , P The result (< 0.001) indicates that spirobroussonin C can effectively relieve the clinical symptoms of atopic dermatitis.

[0081] Regarding skin barrier damage, the TEWL values ​​of each spirobroussonin C dose group increased gradually, and after day 8, the TEWL values ​​of the high-dose and medium-dose groups showed a decreasing trend. Compared with the model group, the TEWL values ​​of each spirobroussonin C dose group were significantly lower. Figure 8 , P < 0.001). This indicates that spirobroussonin C can effectively alleviate skin barrier damage induced by MC903 in a mouse model.

[0082] Prevention and treatment of AD-like skin pathological changes induced by compound spirobroussonin C

[0083] HE staining and toluidine blue staining revealed that MC903-treated mice showed thickened epidermis, dermal hyperplasia, and a significant increase in mast cell count in the ear skin, exhibiting pathological features similar to human Alzheimer's disease (AD). After treatment with spirobroussonin C, the thickness of the mouse ear epidermis and the degree of mast cell infiltration were significantly improved compared to the model group. Figure 9 ).

[0084] Prevention and treatment of MC903-induced inflammatory response in Alzheimer's disease (AD) by compound spirobroussonin C

[0085] Elevated serum IgE levels and type 2 inflammatory response are the main characteristics of atopic dermatitis. For example... Figure 10 As shown, serum IgE levels were significantly reduced after spirobroussonin C intervention ( P < 0.001), showing a dose-dependent effect; high-dose compound treatment significantly reduced the levels of IL-4, IL-13, IL-33, and TSLP in mouse ear tissues compared to the model group, which was statistically significant. This indicates that spirobroussonin C primarily alleviates the inflammatory response in Alzheimer's disease (AD) by decreasing the levels of Th2 cytokines (IL-13, IL-4, IL-33, TSLP) and IgE.

[0086] In summary, animal studies have shown that spirobroussonin C exhibits significant anti-inflammatory and skin barrier repair effects. It may alleviate AD through a synergistic effect of the "Th2 inflammation suppression-skin barrier repair" dual pathway, and no typical side effects similar to those of glucocorticoids, such as skin atrophy and metabolic disorders, were observed within the experimental dosage range.

[0087] Example 5: Preparation method of topical cream

[0088] This embodiment provides two sets of topical preparations for treating atopic dermatitis: 0.1% spirobroussonin C cream and 1% spirobroussonin C cream, which are prepared as oil-in-water (O / W) topical creams by mixing the following raw materials in the indicated weight percentages:

[0089] 0.1% spirobroussonin C cream: 0.1% spirobroussonin C (purity >98%), 20% petrolatum, 5% ethanol, 5% sodium bicarbonate, 1% methylparaben, and 68.9% distilled water.

[0090] 1% spirobroussonin C cream: 1% spirobroussonin C (purity >98%), 20% petrolatum, 5% ethanol, 5% sodium bicarbonate, 1% methylparaben, and 68% distilled water.

[0091] Example 6: Method 2 for preparing topical cream

[0092] This embodiment provides two sets of topical preparations for treating atopic dermatitis: 0.1% spirobroussonin C cream and 1% spirobroussonin C cream, which are prepared as water-in-oil (W / O) topical creams by mixing the following raw materials in the indicated weight percentages:

[0093] 0.1% spirobroussonin C cream: 0.1% spirobroussonin C (purity >98%), 50% petrolatum, 5% glycerin, 3% glyceryl monostearate, 1% methylparaben, and 40.9% distilled water.

[0094] 1% spirobroussonin C cream: 1% spirobroussonin C (purity >98%), 50% petrolatum, 5% glycerin, 3% glyceryl monostearate, 1% methylparaben, and 40% distilled water.

[0095] Example 7 Preparation method of external spray

[0096] 1% spirobroussonin C spray: 1% spirobroussonin C (purity >98%) is dissolved in ethanol, then 1,2-propanediol and purified water are added, stirred thoroughly, filtered, filled and sterilized to prepare the spray.

[0097] Example 8: Preparation method of injection solution

[0098] Take the compound spirobroussonin C, add water for injection as usual, filter, fill and sterilize to prepare an injection solution.

[0099] Example 9: Preparation method of injection

[0100] Take the compound spirobroussonin C, dissolve or suspend it in sterile water for injection, stir well, filter it with a sterile suction funnel, then filter it aseptically, dispense it into ampoules, freeze-dry it at low temperature, and then seal it aseptically to obtain the injection.

[0101] Example 10 Powder Preparation Method

[0102] Take the compound spirobroussonin C and add it to the excipient in a weight ratio of 9:1 to prepare a powder.

[0103] Example 11 Tablet Preparation Method

[0104] Take the compound spirobroussonin C and add it to the excipient in a weight ratio of 1:5 to 1:10, then granulate and compress the mixture into tablets.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A spirovetiverine compound, characterized in that, The structural formula of the compound is as follows: 。 2. The process for the preparation of the spirocyclic crytophan compounds according to claim 1, characterized in that, The method is operated as follows: dry Broussonetia papyrifera aerial parts are taken, crushed, and extracted with an ethanol solution, and the solvent is recovered to obtain an extract, which is dispersed in water, and sequentially extracted with petroleum ether, chloroform, ethyl acetate and n-butanol, wherein the chloroform extraction part is concentrated under reduced pressure, and then subjected to normal phase silica gel column chromatography, and gradient elution is performed with petroleum ether / ethyl acetate as the mobile phase at a ratio of 80:1, 50:1, 20:1, 10:1, 5:1, 1:1, 1:2, and 0:1 to obtain five components Fr. A-Fr. E; The Fr. C part is separated by MCI reverse phase chromatography column, gradient elution is performed with methanol / water as the eluent at a ratio of 70:30, 80:20, 90:10, and 100:0, and 6 components Fr. C-1-Fr. C-6 are obtained by combination; the Fr. C-1 part is separated by Sephadex LH-20 gel column chromatography with methanol elution, and then purified by semi-preparative HPLC, and the RP-C18 chromatographic column is selected, and methanol / water or acetonitrile / water is selected as the mobile phase at a volume ratio of 20-40:80-60 to elute to obtain the spiro Broussonetia papyrifera nins.

3. The method for preparing spirocyclic styracil compounds according to claim 2, characterized in that, The concentration of the ethanol solution is 50%-100% v / v; the amount used is 5-8 times the mass of the Broussonetia papyrifera powder, and extraction is performed 1-4 times.

4. A pharmaceutical composition, characterized by, The composition comprises the compound of claim 1 and / or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

5. The pharmaceutical composition of claim 4, wherein, The composition further comprises other pharmaceutically active ingredients.

6. Use of the compound of claim 1 or the pharmaceutical composition of claim 4 or 5 in the preparation of a drug for preventing and / or treating inflammation.

7. Use of the compound of claim 1 or the pharmaceutical composition of claim 4 or 5 in the preparation of a drug for preventing and / or treating atopic dermatitis.

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