Stem cell factor composite pharmaceutical composition for treating allergic dermatitis and preparation method of stem cell factor composite pharmaceutical composition

Abstract

The invention relates to the field of medicines, in particular to a stem cell factor composite medicine composition for treating allergic dermatitis and a preparation method thereof. The composite pharmaceutical composition is prepared from the following raw materials in parts by weight: 5 to 10 parts of stem cell factors, 2 to 6 parts of agilawood extract, 1 to 3 parts of forskolin and 0.1 to 0.3 part of stabilizer. The pharmaceutical composition can effectively inhibit bacteria, relieve pain and relieve itching, and can remarkably relieve itching and inflammation caused by allergic dermatitis by inhibiting release of inflammatory factors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a stem cell factor complex drug composition for treating allergic skin inflammation and a preparation method thereof. BACKGROUND

[0002] Atopic dermatitis (AD) is a common chronic, relapsing, inflammatory skin disease characterized by intense itching, dry skin, erythema, exudation and crusting, which seriously affects the quality of life of patients. The pathogenesis of the disease is complex, involving multiple factors such as skin barrier dysfunction, immune regulation imbalance (such as excessive activation of Th2 type immune response), microbial community imbalance and environmental allergen triggering. These factors interweave with each other, forming a vicious cycle: the damaged skin barrier makes it easier for allergens to invade, triggering immune inflammation; and the inflammatory response further damages the skin barrier and causes severe itching, scratching behavior aggravating the skin lesions, making the disease persistent.

[0003] Currently, the main treatment for atopic dermatitis relies on glucocorticoids and calcineurin inhibitors and other immunosuppressive drugs. Although these drugs can effectively control acute symptoms, long-term use may lead to skin atrophy, telangiectasia, pigmentation and potential systemic side effects, making patients, especially children, face huge treatment concerns and safety risks. Therefore, there is an urgent need for a new therapy that can break the above vicious cycle, has both repair and treatment effects, and avoids the side effects of hormones. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, one of the purposes of the present application is to provide a stem cell factor complex drug composition for treating allergic skin inflammation, which can effectively inhibit bacteria, relieve pain and itching, and significantly relieve itching and inflammation caused by atopic dermatitis by inhibiting the release of inflammatory factors.

[0005] The second purpose of the present application is to provide a preparation method of a stem cell factor complex drug composition for treating allergic skin inflammation, which is simple in process.

[0006] One of the purposes of the present application is achieved by the following technical solutions: A stem cell factor complex drug composition for treating allergic skin inflammation, comprising the following raw materials by weight: stem cell factor 5-10 parts, chuanxiong extract 2-6 parts, forskolin 1-3 parts, and stabilizer 0.1-0.3 parts.

[0007] Preferably, the stabilizer is schizophyllan.

[0008] Preferably, the preparation method of the schizophyllan is as follows: (1) adding water to the Schizophyllum hyphae for soaking, then reflux extraction, and obtaining a first filtrate and a first residue after filtration; (2) adding water to the first residue for reflux extraction, and obtaining a second filtrate and a second residue after filtration; (3) combining the first filtrate and the second filtrate, vacuum concentration, alcohol precipitation, centrifugation, and drying to obtain the Schizophyllum polysaccharide.

[0009] Preferably, the amount of water added in step (1) is 12-16 times the mass of the Schizophyllum hyphae; the soaking time is 2-4 h; and the reflux extraction time is 1.5-2.5 h.

[0010] Preferably, the amount of water added in step (2) is 8-10 times the mass of the first residue; and the reflux extraction time is 1.5-2.5 h.

[0011] Preferably, in step (3), the vacuum concentration is to 0.05-0.1 times the total volume of the first filtrate and the second filtrate; and the volume fraction of ethanol in the alcohol precipitation is 70-80%.

[0012] Preferably, the stem cell factor is umbilical cord mesenchymal stem cell factor.

[0013] Preferably, the preparation process of the umbilical cord mesenchymal stem cell factor is as follows: The umbilical cord mesenchymal stem cells obtained by primary culture are cultured until the cell fusion degree reaches 80-90%, then subcultured, and the subculture is continued for 6 times; the cell culture solution of P3-P5 generations is collected, filtered and concentrated through a microfiltration membrane and an ultrafiltration membrane, the cut-off liquid is collected, and vacuum freeze-drying is performed.

[0014] The second purpose of the present application is achieved by the following technical solution: The preparation method of the stem cell factor composite pharmaceutical composition for treating allergic skin inflammation comprises the following steps: According to the weight parts, the raw materials are weighed and mixed uniformly.

[0015] Compared with the prior art, the present application has the following advantages: 1. The present application provides a composite pharmaceutical composition comprising stem cell factor, Agarwood extract, Forskolin, and stabilizer. The composite pharmaceutical composition exhibits multiple synergistic effects in treating allergic skin inflammation. Specifically, the stem cell factor can effectively promote skin barrier repair; the Agarwood extract and Forskolin can synergistically inhibit bacteria, relieve pain, and relieve itching, and significantly reduce the itching and inflammatory reaction caused by allergic dermatitis by inhibiting the release of inflammatory factors, thereby achieving good therapeutic effect while avoiding the side effects of traditional hormone therapy.

[0016] 2. The stabilizer of the present invention is Schizophyllum commune polysaccharide, which can effectively enhance the activity and stability of stem cell factors and further enhance their repair effect. On the other hand, by utilizing the good film-forming properties of this polysaccharide, a protective layer can be formed on the inflamed skin wound, which helps to block external stimuli and prevent secondary infection, thereby achieving dual protection for the wound. Attached Figure Description

[0017] Figure 1 This is a morphological diagram of the primary cultured umbilical cord mesenchymal stem cells of this invention. Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments shall be performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0019] The extraction method of agarwood extract in this invention is as follows: Add 8 times the mass of agarwood powder to a 50% ethanol aqueous solution, soak for 24 hours, filter and centrifuge to obtain an extract, and distill to recover water and ethanol to obtain an extract paste. Add 5 times the mass of water to the extract, mix and dissolve evenly, centrifuge to separate, discard the upper oily layer, take the lower water-soluble layer, concentrate and dry, and pulverize to obtain agarwood extract.

[0020] The preparation process of umbilical cord mesenchymal stem cell factors in this invention is as follows: The umbilical cord tissue was cleaned with PBS buffer and then cut into 2mm pieces using surgical scissors. 3 Tissue samples of varying sizes were plated in 10cm culture dishes (serum-free DMEM / F12 medium containing 10% fetal bovine serum) and cultured at 37°C in a 5% CO2 cell culture incubator until cell confluence reached 80%. Cells were then digested with Tryspin-EDTA and harvested to obtain primary cultured umbilical cord mesenchymal stem cells. (See cell morphology diagram for reference.) Figure 1 According to 1×10 4Primary umbilical cord mesenchymal stem cells were seeded at a density of cells / mL in DMEM / F12 medium supplemented with 10% FBS. Once cell confluence reached 90%, 0.25% FBS was used... Cells were digested with EDTA-containing trypsin solution and passaged in a 37°C, 5% CO2 cell culture incubator at a passage ratio of 1:2 for 6 consecutive passages. Cell culture medium from passages P3 to P5 was collected, and the supernatant was concentrated by passing it through a 0.24 μm microfiltration membrane and then through a 50 KD ultrafiltration membrane. The concentrate was then passed through a 3 KD ultrafiltration membrane, and the retentate was collected. A lyophilization protectant was added to the retentate at a volume ratio of 5:1, and the cells were freeze-dried under vacuum at 10 Pa and -70°C for 36 h to obtain the final product. The lyophilization protectant consisted of the following ingredients: 20% (v / v) mannitol, 3% (w / v) dextran, 15% (v / v) propylene glycol, 2% (w / v) methylcellulose, and 60% (v / v) deionized water.

[0021] Preparation Example 1 This preparation example provides a polysaccharide from *Schizophyllum commune*, prepared by the following method: (1) Soak the mycelium of Schizophyllum commune in 15 times the amount of water for 3 hours, then reflux for 2 hours, and filter to obtain the first filtrate and the first filter residue; (2) Add 9 times the amount of water to the first filter residue, reflux for 2 hours, filter to obtain the second filtrate and the second filter residue; (3) Combine the first filtrate and the second filtrate, concentrate them under vacuum to 0.08 times the total volume of the first filtrate and the second filtrate, add 5 times the volume of 75% ethanol aqueous solution, mix well, let stand for 4 hours, centrifuge at 4000 rpm for 12 minutes, discard the supernatant, and dry at 70°C to constant weight to obtain the Schizophyllum polysaccharide.

[0022] Preparation Example 2 This preparation example provides a polysaccharide from *Schizophyllum commune*, prepared by the following method: (1) Soak the mycelium of Schizophyllum commune in 12 times the amount of water for 2 hours, then reflux for 1.5 hours, filter to obtain the first filtrate and the first filter residue; (2) Add 8 times the amount of water to the first filter residue, reflux and extract for 1.5 hours, filter to obtain the second filtrate and the second filter residue; (3) Combine the first filtrate and the second filtrate, concentrate them under vacuum to 0.05 times the total volume of the first filtrate and the second filtrate, add 5 times the volume of 70% ethanol aqueous solution, mix well, let stand for 4 hours, centrifuge at 4000 rpm for 12 minutes, discard the supernatant, dry at 70°C to constant weight, and obtain the Schizophyllum polysaccharide.

[0023] Preparation Example 3 This preparation example provides a polysaccharide from *Schizophyllum commune*, prepared by the following method: (1) Soak the mycelium of Schizophyllum commune in 16 times the amount of water for 4 hours, then reflux for 2.5 hours, filter to obtain the first filtrate and the first filter residue; (2) Add 10 times the amount of water to the first filter residue, reflux and extract for 2.5 hours, filter to obtain the second filtrate and the second filter residue; (3) Combine the first filtrate and the second filtrate, concentrate them under vacuum to 0.1 times the total volume of the first filtrate and the second filtrate, add 5 times the volume of 80% ethanol aqueous solution, mix well, let stand for 4 hours, centrifuge at 4000 rpm for 12 minutes, discard the supernatant, dry at 70°C to constant weight, and obtain the Schizophyllum polysaccharide.

[0024] Example 1 This embodiment provides a stem cell factor compound pharmaceutical composition for treating allergic skin inflammation, comprising the following raw materials in parts by weight: 7 parts stem cell factor, 5 parts agarwood extract, 2 parts forscorin, and 0.2 parts of Schizophyllum commune polysaccharide from Preparation Example 1.

[0025] This embodiment also provides a method for preparing the above-mentioned stem cell factor complex drug composition for treating allergic skin inflammation, including the following steps: Weigh out the ingredients according to the stated weight proportions and mix them thoroughly.

[0026] Example 2 This embodiment provides a stem cell factor compound pharmaceutical composition for treating allergic skin inflammation, comprising the following raw materials in parts by weight: 5 parts stem cell factor, 2 parts agarwood extract, 1 part forscorin, and 0.1 parts of Schizophyllum commune polysaccharide from Preparation Example 2.

[0027] This embodiment also provides a method for preparing the above-mentioned stem cell factor complex drug composition for treating allergic skin inflammation, including the following steps: Weigh out the ingredients according to the stated weight proportions and mix them thoroughly.

[0028] Example 3 This embodiment provides a stem cell factor compound pharmaceutical composition for treating allergic skin inflammation, comprising the following raw materials in parts by weight: 10 parts stem cell factor, 6 parts agarwood extract, 3 parts forscorin, and 0.3 parts polysaccharide of Schizophyllum commune from Preparation Example 3.

[0029] This embodiment also provides a method for preparing the above-mentioned stem cell factor complex drug composition for treating allergic skin inflammation, including the following steps: Weigh out the ingredients according to the stated weight proportions and mix them thoroughly.

[0030] Comparative Example 1 The difference between this comparative example and Example 1 is that the polysaccharide of *Schizophyllum commune* from Preparation Example 1 was not added; otherwise, it is the same as Example 1.

[0031] Comparative Example 2 The difference between this comparative example and Example 1 is that no Qinan agarwood extract was added; otherwise, it is the same as Example 1.

[0032] Comparative Example 3 The difference between this comparative example and Example 1 is that no forscorin was added; otherwise, they are the same as in Example 1.

[0033] Example 1 The stem cell factor compound drug compositions of Examples 1-3 and Comparative Examples 1-3 were stored at 4°C for 6 months. After being taken out, they were re-dissolved in water, and the contents of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in each drug composition were detected using an ELISA kit. The specific results are shown in Table 1.

[0034] Table 1 As shown in Table 1, compared with Comparative Example 1, the drug obtained in Example 1 still had a higher content of stem cell factors after 6 months of storage. These results indicate that the addition of *Schizophyllum commune* polysaccharide can effectively improve the stability of stem cell factors.

[0035] Example 2 2.1 Establishment of a SADBE-induced chronic pruritus model of allergic dermatitis Ninety male C57BL mice, weighing 22-25g, were acclimatized for one week and had their abdominal hair shaved (2cm×2cm) two days before the start of the experiment. Ten mice were randomly selected as the blank control group, and the rest were used as the model group.

[0036] Starting from day 1 of the experiment, sensitization was performed at a fixed time every morning. The model group was sensitized with 25 μL of 1% SADBE acetone solution (prepared fresh each time, 10 mg SADBE dissolved in 1 mL of acetone) on the abdomen, while the control group was sensitized with 25 μL of acetone on the abdomen for 3 consecutive days. After 5 days (day 8), allergic challenge was performed. Two days before challenge (day 6), the neck and back of the mice were shaved (2 cm × 2 cm). Starting from day 8, the model group mice were challenged by topical application of 25 μL of 1% SADBE acetone solution (prepared fresh each time) on the neck and back, while the control group mice were challenged by topical application of 25 μL of acetone on the neck and back for 3 consecutive days. On the morning of day 11, behavioral assessment, skin lesion assessment, and skinfold thickness measurement were performed on the experimental animals to evaluate the success of the modeling.

[0037] 2.2 Grouping Mice that successfully developed the model were randomly divided into 7 groups, with 10 mice in each group. The 7 groups were: model group, Example 1-3 groups, Comparative Example 2-3 groups, and positive control group.

[0038] 2.3 Administration method Model group: Apply water evenly to the shaved area, 200 μL each time, and wipe it off with a sterile alcohol cotton ball soaked in warm water after 30 minutes. Administer the medication 3 times a day, with an interval of 4 hours between each administration, for 7 consecutive days. Blank control group: Water was evenly applied to the shaved area, 200 μL each time. After 30 minutes, it was wiped off with a sterile alcohol cotton ball soaked in warm water. The drug was administered 3 times a day, with an interval of 4 hours between each administration, for 7 consecutive days. Positive control group: Hydrocortisone acetate propyl ester was evenly applied to the shaved area, 100 μL each time, without wiping, once a day for 7 consecutive days; Example 1 group: The drug composition of Example 1 was evenly applied to the shaved area, with a dosage of 0.1 g / cm² per application. -2 After 30 minutes, wipe it off with a sterile alcohol-soaked cotton ball dipped in warm water. Administer the medication 3 times a day, with a 4-hour interval between each administration, for 7 consecutive days. Example 2 group: The drug composition of Example 2 was evenly applied to the shaved area, with a dosage of 0.1 g / cm³ per application. -2 After 30 minutes, wipe it off with a sterile alcohol-soaked cotton ball dipped in warm water. Administer the medication 3 times a day, with a 4-hour interval between each administration, for 7 consecutive days. Example 3 group: The drug composition of Example 3 was evenly applied to the shaved area, with a dosage of 0.1 g / cm³ per application. -2 After 30 minutes, wipe it off with a sterile alcohol-soaked cotton ball dipped in warm water. Administer the medication 3 times a day, with a 4-hour interval between each administration, for 7 consecutive days. Comparative Example 2: The drug composition of Comparative Example 2 was evenly applied to the shaved area, with a dosage of 0.1 g / cm² per application. -2 After 30 minutes, wipe it off with a sterile alcohol-soaked cotton ball dipped in warm water. Administer the medication 3 times a day, with a 4-hour interval between each administration, for 7 consecutive days. Comparative Example 3: The drug composition of Comparative Example 3 was evenly applied to the shaved area, with a dosage of 0.1 g / cm³ per application. -2 After 30 minutes, wipe it off with a sterile alcohol-soaked cotton ball dipped in warm water. Administer the medication three times a day, with a 4-hour interval between each administration, for a total of 7 days.

[0039] 2.4 Detection Indicators 2.4.1 Results of Mouse Scratching Behavior After 7 days of continuous drug administration, mice in each group were placed in transparent observation boxes, with white paper separating the boxes. Videos were taken from directly above, and scratching activity was counted over 1 hour. The scratching count was defined as the period from when the mouse's hind paw was raised to the area of ​​the body to be scratched (the neck and back model area) until the hind paw was placed back on the ground. One round of scratching may last a few seconds. Due to the rapid speed of a single scratch, only the number of rounds was recorded. The results are shown in Table 2.

[0040] 2.4.2 Results of Skinfold Thickness Variation After 7 days of continuous administration, the thickness of the skinfold on the back of the neck of the mice was measured with calipers. Each mouse was measured 3 times and the average value was taken. The results are shown in Table 2.

[0041] 2.4.3 Results of changes in mRNA expression of inflammatory factors Skin lesions on the back of the neck of mice in each group were collected and ground, and total RNA was extracted using the TransZol Up Plus RNA Kit. cDNA was obtained by reverse transcription using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit. Real-time PCR was performed on the obtained cDNA, with GAPDH selected as an internal reference gene, to determine the expression of target gene mRNAs such as IL-1β, TNF-α, CXCL10, and Clc20 in the skin. A 10 μL real-time PCR reaction system was established according to the manufacturer's instructions. Melting curves were plotted for real-time PCR to detect the specificity of the amplified genes. The experimental data were analyzed using a 2-1... -ΔΔCt The analysis and statistics were performed using the methods described. Primer sequences are shown in Table 3, and experimental results are shown in Table 4.

[0042] Table 2 Table 3 Table 4 As shown in Table 2, the pharmaceutical compositions of Examples 1-3 and Comparative Examples 2-3 were all effective in alleviating pruritus and skin thickening in the SADBE-induced mouse model. However, the effects of Comparative Examples 2-3 were slightly less pronounced than those of Example 1.

[0043] As shown in Table 3, the drug compositions used in Examples 1-3 and Comparative Examples 2-3 were able to reduce the expression of inflammatory factor mRNAs such as TNF-α, IL-1β, CXCL10, and Clc20. However, the effects of Comparative Examples 2-3 were slightly weaker than those in Example 1.

[0044] Analysis of the experimental data in Tables 2-3 shows that the combined use of Qinan agarwood extract and foscolin exhibits a synergistic effect, effectively inhibiting bacteria, relieving pain and itching, and significantly alleviating itching and inflammation caused by allergic dermatitis by inhibiting the release of inflammatory factors. This combined therapy has a clear therapeutic effect and avoids the side effects of hormonal drugs.

[0045] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A stem cell factor complex pharmaceutical composition for treating allergic skin inflammation, characterized in that, The ingredients include the following parts by weight: 5-10 parts stem cell factor, 2-6 parts agarwood extract, 1-3 parts foscolin, and 0.1-0.3 parts stabilizer.

2. The stem cell factor compound pharmaceutical composition for treating allergic skin inflammation according to claim 1, characterized in that, The stabilizer is Schizophyllum commune polysaccharide.

3. The stem cell factor compound pharmaceutical composition for treating allergic skin inflammation according to claim 2, characterized in that, The preparation method of the *Schizophyllum commune* polysaccharide is as follows: (1) Soak the mycelium of Schizophyllum commune in water, then reflux extract, filter to obtain the first filtrate and the first filter residue; (2) Add water to the first filter residue and reflux extract, then filter to obtain the second filtrate and the second filter residue; (3) Combine the first filtrate and the second filtrate, and obtain the Schizophyllum polysaccharide by vacuum concentration, alcohol precipitation, centrifugation and drying.

4. The stem cell factor compound pharmaceutical composition for treating allergic skin inflammation according to claim 2, characterized in that, The amount of water added in step (1) is 12-16 times the mass of the mycelium of Schizophyllum commune; the soaking time is 2-4 hours; and the reflux extraction time is 1.5-2.5 hours.

5. The stem cell factor compound pharmaceutical composition for treating allergic skin inflammation according to claim 2, characterized in that, The amount of water added in step (2) is 8-10 times the mass of the first filter residue; the reflux extraction time is 1.5-2.5h.

6. The stem cell factor complex pharmaceutical composition for treating allergic dermatitis according to claim 2, characterized in that, In step (3), the vacuum concentration is carried out to 0.05-0.1 times the total volume of the first and second filtrates; the volume fraction of ethanol in the alcohol precipitation is 70-80%.

7. The stem cell factor complex pharmaceutical composition for treating allergic dermatitis according to claim 1, characterized in that, The stem cell factor is umbilical cord mesenchymal stem cell factor.

8. The stem cell factor complex pharmaceutical composition for treating allergic dermatitis according to claim 7, characterized in that, The preparation process of the umbilical cord mesenchymal stem cell factor is as follows: The umbilical cord mesenchymal stem cells obtained from primary culture are cultured until the cell confluence reaches 80-90%, and then passaged for 6 consecutive times. The cell culture medium from P3 to P5 is collected, filtered and concentrated through microfiltration and ultrafiltration membranes, the retentate is collected, and then freeze-dried under vacuum.

9. A method for preparing a stem cell factor complex pharmaceutical composition for treating allergic dermatitis according to any one of claims 1-8, characterized in that, Includes the following steps: Weigh out the ingredients according to the stated weight proportions and mix them thoroughly.

Images