Kushenin composition and application thereof

Abstract

The invention relates to the technical field of medicines. The invention provides a kushenin composition and application thereof. The kushenin composition is prepared from the following raw materials in parts by mole: 1 to 10 parts of kushenin and 1 to 10 parts of levistilide A. The kushenin composition is prepared from the following raw materials in parts by mole; when the composition is applied to HaCaT cells, compared with single use of kushenin, proliferation of keratinocytes can be more remarkably inhibited, and generation of inflammatory factors can be reduced. In a psoriasis-like mouse model, compared with the single use of the kushenin, the composition can more effectively improve the psoriasis symptom of the mouse and obviously enhance the treatment effect of the kushenin on the psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a matrine composition and application thereof. Background Art

[0002] Psoriasis, commonly known as psoriasis, is a chronic skin disease mediated by environmental factors, polygenic inheritance and immunity. Its clinical manifestations are scaly erythema or plaques, and some patients also have joint symptoms and nail abnormalities. Psoriasis not only affects the patient's quality of life, but is also associated with the occurrence of metabolic syndrome, cardiovascular disease and other diseases, and may be life-threatening in severe cases.

[0003] Oxymatrine (OMT) is a natural alkaloid extracted from Chinese herbs such as Sophora flavescens and Sophora flavescens. 15 H 24 N2O2. Studies have shown that matrine has a wide range of pharmacological activities, including anti-tumor, anti-fibrosis, anti-viral and anti-inflammatory effects. Many literature reports have shown that matrine has certain potential in the treatment of psoriasis. For example, LiN et al. found that matrine can inhibit the proliferation of keratinocytes in BALB / c mice with psoriatic dermatitis induced by imiquimod, reduce the infiltration of PCNA+ and CD3+ cells in the lesions, and reduce the expression of MyD88 protein and inflammatory factors IL-12, IL-23, and IL-1β, thereby alleviating psoriatic dermatitis. Jiang WW et al. also showed that matrine can inhibit the proliferation of HaCaT cells in a dose- and time-dependent manner, and when used in combination with acitretin, it induces cell cycle arrest and autophagy by activating the PI3K / Akt / mTOR pathway, producing a synergistic effect. However, most of the current studies focus on the effects of a single ingredient, and lack the exploration of the combination of matrine with other active ingredients to enhance its therapeutic effect. Summary of the invention

[0004] The purpose of the present invention is to provide a matrine composition and application thereof, which significantly enhances the effect of matrine in treating psoriasis by adding compound ingredients.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a matrine composition, which comprises the following raw materials in molar parts: 1-10 parts of matrine and 1-10 parts of angelica lactone A.

[0007] Preferably, the composition comprises the following raw materials in molar proportions: 2 to 8 parts of matrine and 2 to 8 parts of angelica lactone A.

[0008] Preferably, the composition comprises the following raw materials in molar proportions: 3-7 parts of matrine and 3-7 parts of angelica lactone A.

[0009] Preferably, the composition comprises the following raw materials in molar proportions: 4-6 parts of matrine and 4-5 parts of angelica lactone A.

[0010] The present invention also provides application of the composition in preparing a medicine for preventing or treating psoriasis.

[0011] The present invention also provides a medicine for preventing or treating psoriasis, wherein the composition contains the composition.

[0012] Preferably, the drug is in the form of an injection.

[0013] The present invention provides a matrine composition and its application, wherein the composition comprises the following raw materials in molar parts: 1 to 10 parts of matrine and 1 to 10 parts of angelica lactone A. When the composition of the present invention is applied to HaCaT cells, it can more significantly inhibit the proliferation of keratinocytes and reduce the production of inflammatory factors compared to the use of matrine alone. In a psoriasis-like mouse model, compared to the use of matrine alone, the composition can more effectively improve the psoriasis symptoms of mice and significantly enhance the therapeutic effect of matrine on psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : The effect of matrine on HaCaT cells induced by imiquimod, wherein A is the proliferation promoting effect of different concentrations of imiquimod on HaCaT cells; wherein B is the effect of matrine on HaCaT cells induced by imiquimod.

[0015] Figure 2 The figure shows the effect of angelica lactone A on the inhibitory effect of matrine on HaCaT cell proliferation, where A is the structure of angelica lactone A; B is the CCK-8 assay showing the change in the activity of HaCaT after the addition of angelica lactone A; and C is the Giemsa staining showing the change in the number of HaCaT after the addition of angelica lactone A.

[0016] Figure 3 This is the effect of angelica lactone A on the inhibition of oxymatrine on the secretion of inflammatory factors by HaCaT cells, where A represents the change of inflammatory factor IL-17; B represents the change of inflammatory factor IL-23.

[0017] Figure 4 This is the effect of angelica lactone A on matrine in improving the symptoms of psoriasis mice, where A is the change in the appearance of the back of psoriasis mice; and B is HE staining of the skin. DETAILED DESCRIPTION

[0018] The invention provides a matrine composition, which comprises the following raw materials in molar parts: 1-10 parts of matrine and 1-10 parts of angelica lactone A.

[0019] In the present invention, the composition preferably comprises the following raw materials in molar proportions: 2 to 8 parts of matrine and 2 to 8 parts of angelica lactone A.

[0020] In the present invention, the composition preferably comprises the following raw materials in molar proportions: 3 to 7 parts of matrine and 3 to 7 parts of angelica lactone A.

[0021] In the present invention, the composition preferably comprises the following raw materials in molar proportions: 4-6 parts of matrine and 4-5 parts of angelica lactone A.

[0022] The present invention also provides application of the composition in preparing a medicine for preventing or treating psoriasis.

[0023] The present invention also provides a medicine for preventing or treating psoriasis, wherein the composition contains the composition.

[0024] In the present invention, the dosage form of the drug is an injection.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example

[0027] 1. Cell culture:

[0028] HaCaT cells (China Center for Type Culture Collection, Wuhan) were cultured at 37°C and 5% CO2 in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0029] 2. CCK-8 cell activity assay:

[0030] Cell counting: When HaCaT cells reach a monolayer covering the bottom of the culture dish, use trypsin to digest the cells for 10 minutes. Then add 2 μL of trypan blue dye and mix with 18 μL of cell suspension for staining. Count the cells using a cell counter and adjust the cell concentration to 6×10 4 / mL.

[0031] Cell plating: After counting, the cell suspension was inoculated into a 96-well plate at a concentration of 6000 cells / well. PBS was added to the surrounding wells to reduce the edge effect. After standing for 5 minutes, the plates were transferred to a CO2 incubator for further culture.

[0032] Imiquimod modeling: After the cells adhered for 12 hours, imiquimod solution was prepared at concentrations of 3.6μg / mL, 7.8μg / mL, 15.6μg / mL, 31.2μg / mL, 62.5μg / mL, and 125μg / mL. After removing the upper culture medium from each well, 100μL of culture medium containing imiquimod was added, and only complete culture medium was added to the blank control group. Six replicate wells were set up for each group and transferred to a CO2 incubator for continued culture for 24 hours.

[0033] Matrine treatment: After the cells adhered to the wall, matrine treatment solutions with concentrations ranging from 7.5mM to 45mM were added. The model group was added with culture solution containing imiquimod and cultured for 24 hours.

[0034] Treatment with matrine + angelica lactone A: After adhesion, different concentrations of angelica lactone A (2, 4, 6 portions) and 7.5 mM matrine were added. Six replicate wells were set up in each group and transferred to a CO2 incubator for further culture for 24 hours.

[0035] Cell viability detection: After administration, the culture medium was removed, 100 μL of 10% Cell Counting Kit-8 solution was added, incubated for 40 minutes, and the absorbance value was measured at 450 nm using an ELISA reader.

[0036] 3. Giemsa staining:

[0037] HaCaT cells were seeded in 6-well plates (2×10 5 / well), and after the cells adhered to the wall, imiquimod, 7.5mM matrine, and 2, 4, and 6 concentrations of angelica lactone A were added. After 24 hours, the culture medium was removed, washed with PBS, and fixed with 1mL methanol for 5 minutes after drying. The methanol was removed again, and 1mL Giemsa stain was added for 30 minutes. The stain was diluted with PBS (1× concentration), washed four times with water after staining, and photographed.

[0038] 4. Determination of inflammatory factors IL-17 and IL-23

[0039] Take out the kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. According to the number of samples and standards, prepare the required solutions and determine the number of strips. Add 100 μL of sample or standard solution to each well (only add standard or sample diluent to the blank control well), seal the reaction well with sealing tape, and incubate in a 37°C incubator for 90 minutes (except for the blank control well). After the incubation, shake off the liquid in the well, add 350 μL of washing solution to each well, let it stand for 30 seconds, shake off the liquid, and pat dry on absorbent paper, repeat 4 times. Then add biotinylated antibody working solution (100 μL / well), incubate at 37°C for 60 minutes, and wash again 4 times. Then add enzyme conjugate working solution (100 μL / well), incubate at 37°C for 30 minutes, and wash 4 times. Finally, add the colorimetric agent (100 μL / well), incubate in the dark for 20 minutes, then add the stop solution (100 μL / well), mix well and quickly measure the OD value at 450 nm (within 5 minutes).

[0040] 5. Psoriasis mouse modeling and drug administration

[0041] All mice were housed in the GLP (Good Laboratory Practice) animal room of Lanzhou University. 50 mice were randomly divided into blank control group, model group, methotrexate group, matrine group, and matrine + angelica lactone A group, with 10 mice in each group.

[0042] Hair was removed from the 2cm×3cm area on the back of mice, and 62.5mg vaseline was applied to the control group, 62.5mg imiquimod cream was applied to the model group, and methotrexate (7mg / kg), oxymatrine (25mg / kg), and oxymatrine + angelica lactone A (25mg / kg + 6 portions) were given to the treatment group on the basis of the model group. Imiquimod was applied every morning, and the corresponding drug treatment was given in the afternoon for a total of 7 days. After the end, photos were taken and samples were taken for HE staining (completed by Wuhan Sewell Company).

[0043] result

[0044] 1. Inhibitory effect of matrine on imiquimod-induced proliferation of HaCaT cells

[0045] The results of CCK-8 experiments showed that compared with the blank control group, the viability of HaCaT cells treated with imiquimod (IMQ) increased significantly, and different concentrations of imiquimod showed an increasing trend, among which the proliferation-promoting effect at concentrations of 3.6 μg / mL and 7.2 μg / mL was the most significant (see Figure 1 A), indicating that the HaCaT cell proliferation model was successfully established. Oxymatrine at a concentration of 7.5-45 mM can significantly inhibit the proliferation-promoting effect of imiquimod on HaCaT cells. Within the concentration range studied, the increase in the concentration of oxymatrine and the inhibitory effect on HaCaT cell proliferation showed a significant dose-dependent relationship (see Figure 1 B). Through curve fitting analysis, the IC50 value of matrine for inhibiting the proliferation of HaCaT cells was obtained to be 18.6 mM.

[0046] 2. Enhancement of the inhibitory effect of oxymatrine on the proliferation of HaCaT cells by angelica lactone A

[0047] In this example, the concentration of oxymatrine was 7.5 mM as the basic concentration for subsequent studies. The experimental results showed that when different amounts of oxymatrine A (LeA) were added, namely 2, 4, 6 and 8 parts, compared with the use of 7.5 mM oxymatrine alone, the inhibitory effect of oxymatrine on HaCaT cells was gradually enhanced, and with the increase of the concentration of oxymatrine A, its enhancement effect became more significant (see Figure 2 B). When the amount of angelica lactone A is 4 parts, the IC50 value of oxymatrine can be reached. Compared with the 18.6mM of oxymatrine used alone, the concentration of oxymatrine can be reduced by 11mM. In addition, the Giemsa staining results further confirmed that oxymatrine can inhibit the proliferation of HaCaT cells, and the inhibitory effect on HaCaT cells is more obvious after adding angelica lactone A (see Figure 2 C), With the addition of angelica lactone A, the number of HaCaT cells gradually decreased.

[0048] 3. The enhancing effect of angelica lactone A on the inhibition of inflammatory response of HaCaT cells by matrine

[0049] In this example, a concentration of 7.5 mM of matrine was selected for further study. The results showed that matrine could significantly reduce the levels of inflammatory factors IL-17 and IL-23 in HaCaT cells induced by imiquimod. After adding different amounts of angelica lactone A, the ability of matrine to reduce the level of inflammatory factors was further enhanced, and there was a significant difference (see Figure 3 ).

[0050] 4. Angelica lactone A enhances the effect of matrine on improving psoriasis-like mice

[0051] In animal experiments, compared with the blank group, mice in the imiquimod-treated group showed obvious scales on their backs and thickened keratin, with typical psoriasis symptoms. The positive control drug methotrexate (MTX) can significantly reduce the number of scales and thin the stratum corneum, and matrine also showed a similar effect, significantly reducing the number of scales and the thickness of the stratum corneum. The combination of matrine and angelica lactone A showed a more obvious effect than the matrine alone group, with the back skin morphology close to the blank group, with almost no scales (see Figure 4 HE staining results showed that matrine could effectively reduce the thickness of the mouse back skin, and when used in combination with angelica lactone A, the effect of reducing skin thickness was more significant (see Figure 4B). In conclusion, angelica lactone A can significantly enhance the improvement effect of matrine on psoriasis-like mice.

[0052] It can be seen from the above embodiments that the present invention provides a matrine composition and its application, wherein the composition comprises the following raw materials in molar parts: 1 to 10 parts of matrine and 1 to 10 parts of angelica lactone A. When the composition of the present invention is applied to HaCaT cells, it can more significantly inhibit the proliferation of keratinocytes and reduce the production of inflammatory factors compared to the use of matrine alone. In a psoriasis-like mouse model, compared to the use of matrine alone, the composition can more effectively improve the psoriasis symptoms of mice and significantly enhance the therapeutic effect of matrine on psoriasis.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A matrine composition, characterized in that: The composition comprises the following raw materials in molar proportions: 1 to 10 parts of matrine and 1 to 10 parts of angelica lactone A.

2. The composition according to claim 1, characterized in that The composition comprises the following raw materials in molar proportions: 2 to 8 parts of matrine and 2 to 8 parts of angelica lactone A.

3. The composition according to claim 2, characterized in that The composition comprises the following raw materials in molar proportions: 3-7 parts of matrine and 3-7 parts of angelica lactone A.

4. The composition according to claim 3, characterized in that The composition comprises the following raw materials in molar proportions: 4-6 parts of matrine and 4-5 parts of angelica lactone A.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a medicament for preventing or treating psoriasis.

6. A drug for preventing or treating psoriasis, characterized in that: The medicine contains the composition according to any one of claims 1 to 4.

7. The drug according to claim 6, characterized in that The dosage form of the drug is injection.

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