Application of 8-debenzoylpaeoniflorin in the preparation of a drug for treating asthma

Abstract

The present invention discloses the use of 8 - debenzoylpaeoniflorin in the preparation of a medicament for treating asthma, and relates to the technical field of traditional Chinese medicine. The present invention provides the use of 8 - debenzoylpaeoniflorin in the preparation of a medicament for treating asthma. The 8 - debenzoylpaeoniflorin has low toxicity, and 8 - debenzoylpaeoniflorin has stronger anti - allergic asthma activity than paeoniflorin at the same dose, and can be applied to the preparation of a medicament for treating allergic asthma.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to the application of 8-desbenzoylpaeoniflorin in the preparation of a drug for treating asthma. Background Art

[0002] Allergic diseases, as a type of chronic disease, seriously affect the work efficiency of patients, reduce their productivity, and thus lead to an increase in social costs. Therefore, the effective and safe treatment of allergic diseases is one of the major challenges faced by public health. An allergic reaction is the most urgent and potentially severe manifestation of allergic diseases. It refers to a physiological change mainly characterized by disorders of the body's physiological functions or tissue cell damage that occurs when the body is re-exposed to the same antigenic stimulus after the initial response to an antigenic substance. According to a report by the World Allergy Organization, 30% - 40% of the world's population suffers from allergic symptoms; due to the lack of severe life-threatening symptoms in the early and middle stages of allergic reactions, these data are often underestimated. Most of the currently available anti-allergy drugs have serious adverse reactions, such as arrhythmia, mental dysfunction, gastrointestinal disorders, and infections. Therefore, it is urgent to explore new effective treatment options. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of 8-desbenzoylpaeoniflorin in the preparation of a drug for treating asthma, and 8-desbenzoylpaeoniflorin exhibits strong anti-allergic asthma activity.

[0004] The present invention provides the application of 8-desbenzoylpaeoniflorin in the preparation of a drug for treating asthma.

[0005] Preferably, the asthma includes allergic asthma.

[0006] Preferably, the therapeutic effect of the drug is manifested as at least one of the following: (1) reducing the expression of inflammatory factors at the diseased site;

[0007] (2) reducing pulmonary inflammatory infiltration;

[0008] (3) reducing the total number of monocytes, the number of eosinophils, and the number of neutrophils.

[0009] Preferably, the inflammatory factors include one or more of IL-6, IL-17A, Tryptase, MMP13, and MPO.

[0010] Preferably, the drug has 8-desbenzoylpaeoniflorin as the sole active ingredient.

[0011] Preferably, the drug has 8-desbenzoylpaeoniflorin and other drugs with therapeutic effects on allergic asthma as co-active ingredients.

[0012] The present invention provides a drug for treating allergic asthma, which uses 8-desbenzoylpaeoniflorin as the active ingredient and also includes pharmaceutically acceptable excipients.

[0013] Preferably, the dosage of the drug is not less than 10 mg / kg based on 8-desbenzoylpaeoniflorin.

[0014] Preferably, 8-desbenzoylpaeoniflorin is the sole active ingredient.

[0015] Preferably, 8-desbenzoylpaeoniflorin and other drugs having a therapeutic effect on allergic asthma are used as the active ingredients together.

[0016] Beneficial effects: The present invention provides the application of 8-desbenzoylpaeoniflorin in the preparation of drugs for treating asthma. The toxicity of 8-desbenzoylpaeoniflorin was evaluated by the MTT method, and it had no effect on the activity of BEAS-2B cells at a concentration lower than 25 μM. An OVA-induced allergic asthma mouse model was constructed to verify the therapeutic effect of 8-desbenzoylpaeoniflorin. The results showed that the total number of monocytes, eosinophils, and neutrophils in the BALF of OVA-sensitized mice were significantly reduced, and it was dose-dependent. The expressions of IL-6, IL-17A, Tryptase, MMP13, and MPO in the lung tissues of allergic asthma mice were reduced, and it was dose-dependent, which confirmed that 8-desbenzoylpaeoniflorin had stronger anti-allergic asthma activity than paeoniflorin at the same dose. Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is a graph showing the pharmacodynamic comparison results of paeoniflorin (PF) and 8-desbenzoylpaeoniflorin (8M) in intervening allergic asthma; A: The results of Wright-Giemsa staining of bronchoalveolar lavage fluid and H&E staining of lung tissues; B: The effects of 8-desbenzoylpaeoniflorin and paeoniflorin on the total number of monocytes, eosinophils, and neutrophils in the BALF of OVA-sensitized mice; C: The results of asthma inflammation scores; The data were analyzed by one-way ANOVA and expressed as mean ± standard deviation. *P<0.05 was statistically significant. *Compared with the control group, *P<0.05, **P<0.01, ***P<0.001, #Compared with the model group, #P<0.05, ##P<0.01, P<0.001, the same below;

[0019] Figure 2 Effects of 8 - debenzoylpaeoniflorin (8M) and paeoniflorin (PF) on the expression of IL - 6, IL - 17A, Tryptase, MMP13 and MPO in the lung tissues of allergic asthma mice; A: Protein blot bands of IL - 6, IL - 17A, Tryptase, MMP13, MPO and GAPDH, B: Analysis of the gray values of protein blot bands;

[0020] Figure 3 Results of the pharmacodynamic comparison of high, medium and low doses of 8 - debenzoylpaeoniflorin (8L, 8M and 8H) in intervening allergic asthma; A: Results of Wright - Giemsa staining of bronchoalveolar lavage fluid and H&E staining of lung tissues; B: Effects of each group of 8 - debenzoylpaeoniflorin on the total number of monocytes, eosinophils and neutrophils in BALF of OVA - sensitized mice; C: Results of asthma inflammation score;

[0021] Figure 4 Results of the effects of 8 - debenzoylpaeoniflorin (8L, 8M, 8H) on the expression of IL - 6, MPO, MMP13 and Tryptase in the lung tissues of allergic asthma mice; A: Protein blot bands of IL - 6, MPO, MMP13, Tryptase and GAPDH, B: Analysis of the gray values of protein blot bands;

[0022] Figure 5 Results of the toxicity evaluation of 8 - debenzoylpaeoniflorin by MTT method. Detailed implementation mode

[0023] The present invention provides the application of 8 - debenzoylpaeoniflorin in the preparation of a drug for treating asthma.

[0024] The asthma described in the present invention preferably includes allergic asthma. In the examples, an OVA - sensitized model mouse is preferably used for verification to confirm the anti - allergic asthma activity of 8 - debenzoylpaeoniflorin (CAS Number: 23532 - 11 - 8). The present invention does not have special limitations on the source of the 8 - debenzoylpaeoniflorin. In the examples, 8 - debenzoylpaeoniflorin purchased from Chengdu Deste Biotechnology Co., Ltd. is used as an example for illustration, but it cannot be considered as the entire protection scope of the present invention only.

[0025] The therapeutic effect of the drug described in the present invention preferably shows at least one of the following: (1) Reducing the expression of inflammatory factors in the diseased part; the inflammatory factors preferably include one or more of IL - 6, IL - 17A, Tryptase, MMP13 and MPO;

[0026] (2) Reducing lung inflammation infiltration;

[0027] (3) Reduce the total number of monocytes, the number of eosinophils, and the number of neutrophils.

[0028] The drug of the present invention preferably uses 8-debenzoylpaeoniflorin as the sole active ingredient, or may use 8-debenzoylpaeoniflorin and other drugs having a therapeutic effect on allergic asthma as active ingredients. The present invention does not limit the types and sources of other drugs having a therapeutic effect on allergic asthma, and common drugs having a therapeutic effect on allergic asthma in the art can be used.

[0029] The present invention provides a drug for treating allergic asthma, which uses 8-debenzoylpaeoniflorin as an active ingredient and also includes pharmaceutically acceptable excipients.

[0030] The drug of the present invention is preferably the same as above and will not be described herein again. The dosage of the drug of the present invention is calculated based on 8-debenzoylpaeoniflorin, and is preferably not less than 10 mg / kg.

[0031] In order to further illustrate the present invention, the application of 8-debenzoylpaeoniflorin provided by the present invention in the preparation of drugs for treating asthma will be described in detail below with reference to the drawings and examples, but they cannot be understood as limiting the protection scope of the present invention.

[0032] Example 1

[0033] 1.1 Drug preparation

[0034] Accurately weigh the powders of paeoniflorin and 8-debenzoylpaeoniflorin, dissolve them in 0.10% CMC-Na solution to prepare a dosing solution (the dosing dose of paeoniflorin is 20 mg / kg; the dosing doses of 8-debenzoylpaeoniflorin are 50 mg / kg, 20 mg / kg, and 10 mg / kg respectively), and store it in a refrigerator at 4°C for later use.

[0035] 1.2 Model establishment and drug administration

[0036] 70 female BALB / c mice, weighing 17 - 22 g, were randomly divided into 7 groups. After adapting to the environment for one week, subsequent experiments were carried out. The experimental groups were divided into a blank group, a model group (OVA sensitization), a paeoniflorin group (PF group 20 mg / kg); a high-dose group of 8-debenzoylpaeoniflorin (8H group 50 mg / kg), a medium-dose group of 8-debenzoylpaeoniflorin (8M group 20 mg / kg), and a low-dose group of 8-debenzoylpaeoniflorin (8L group 10 mg / kg).

[0037] On the 1st day and the 14th day, mice were given a suspension containing 40 μg OVA and 2 mg Al(OH)₃, and the blank group was treated with an equal amount of normal saline. From the 28th day to the 34th day, except for the blank group and the model group, each group was given drug treatment every day. The blank group and the model group were treated with an equal amount of normal saline for 7 days. After drug administration every day, except for the blank group, each group was nebulized and challenged with 5% OVA for 30 min using a nebulizer. The blank group was nebulized with an equal amount of CMC-Na solution containing normal saline for 30 min. The drug administration experiment was carried out by a single intraperitoneal gavage method. After gavage, the diet and drinking water were normal. After the last drug administration, all groups of mice were fasted (water was not prohibited).

[0038] 1.3 Collection of tissues, sera, and BALF

[0039] Twenty-four hours after the last drug administration and OVA nebulization challenge, the mice were anesthetized and blood was collected from the heart. The mouse blood was collected in a centrifuge tube and centrifuged at 3000 rpm for 10 min. After standing and separating, the sera were obtained and stored in a -80 °C refrigerator for later use. The BALB / c female mice were dissected. The mouse lungs were accurately located, the right lung was ligated, and the left lung was rinsed 3 times with 0.4 mL of phosphate buffer solution (PBS). The BALF was collected, dropped on a glass slide, and dried for later use. The mouse lungs were precisely dissected, the largest lobe of the lung was excised, fixed with 10% formalin, and the remaining part was stored in a -80 °C refrigerator for later use in subsequent related experiments.

[0040] 1.4 Cell staining and counting in mouse BALF (Wright-Giemsa staining)

[0041] For the dried glass slides of BALF, the Wright-Giemsa staining method was used to stain the cells on the glass slides. On the glass slides, 7 - 8 drops of staining solution were added and allowed to stand for about 1 min. Then, 10 - 15 drops of pure water were added and mixed well, and stained for about 15 min. After completion, the slides were continuously rinsed with pure water. Finally, the total number of monocytes, the number of eosinophils, and the number of neutrophils were observed under an optical microscope.

[0042] 1.5 Histopathological study of inflammatory infiltration in mouse lungs (H&E staining)

[0043] After the mouse lung tissue samples were fixed with formalin for one week, they were taken out and rinsed with running water to remove the excess formalin on the tissue surface. Tissue samples with a volume of about 1 cm 3The lung tissues were continuously rinsed with flowing tap water for about 2 hours or more. All samples were dehydrated using a fully automatic dehydrator. Subsequently, a tissue embedding machine was used to embed the tissues in paraffin. After cooling to room temperature, all samples were sectioned to a thickness of approximately 4 μm, and then placed on glass slides to make sections. The paraffin sections were placed in an oven and melted at 60 °C for 2 hours or more. After dewaxing, hydration and other operations, H&E staining was performed. After completion, 1-2 drops of neutral gum were dropped on the glass slide, and a coverslip was used to cover the slide, which was then placed under an optical microscope to observe the inflammatory infiltration in the mouse lung tissues.

[0044] In the H&E stained sections, the pathological sections of the mice were scored for asthma inflammation according to the degree of inflammatory infiltration. The scoring range was 1-4 points: 1 point indicated no obvious inflammatory cell infiltration in the tissue and the organ structure was intact; 2 points indicated that the organ structure was intact, but there was a small amount of inflammatory cell infiltration; 4 points indicated damage to the organ wall or epithelial shedding, accompanied by a large number of inflammatory cell infiltrations; 3 points indicated that the degree of inflammatory infiltration was between 2-4 points.

[0045] The results were as Figure 1 shown. Wright-Giemsa staining of bronchoalveolar lavage fluid showed that, compared with the blank group, the total numbers of monocytes, eosinophils and neutrophils in the OVA group mice increased significantly after sensitization. Compared with the OVA-induced model group, both the PF and 8M groups could significantly reduce the release of these cells, and the pharmacological effect of 8M was stronger than that of PF ( Figure 1 in A, B). H&E staining showed that, compared with the blank group, the bronchi of the lung tissues in the OVA group were deeply infiltrated by inflammatory cells. Both the PF and 8M groups could improve the infiltration of inflammatory cells in the OVA-induced mouse lung tissues, and the pharmacological effect of 8M was stronger than that of PFH ( Figure 1 in A). In summary, it was confirmed that 8-debenzoylpaeoniflorin had stronger anti-allergic asthma activity than paeoniflorin at the same dose.

[0046] The results were as Figure 3 shown. Wright-Giemsa staining of bronchoalveolar lavage fluid showed that, compared with the blank group, the total numbers of monocytes, eosinophils and neutrophils in the OVA group mice increased significantly after sensitization. Different doses of 8-debenzoylpaeoniflorin (8L, 8M, 8H) all had a certain degree of effect on reducing the number of white blood cells and showed a certain drug dose-dependence ( Figure 3 in A, B); H&E staining showed that, compared with the blank group, the bronchi of the lung tissues in the OVA group were deeply infiltrated by inflammatory cells. Different doses of 8-debenzoylpaeoniflorin could all improve the infiltration of inflammatory cells in the OVA-induced mouse lung tissues and showed a certain drug dose-dependence ( Figure 3 in A).

[0047] 1.6 Detection of the expression of inflammatory factors and other proteins in mouse lung tissue by Western Blotting

[0048] Take the fresh lung tissue of each group of mice, accurately weigh about 0.1 g of tissue samples, and add 1×PBS buffer containing 1% PMSF, about 5 times the mass volume. After cutting the tissue into pieces, homogenize it with a tissue homogenizer to obtain lung tissue homogenate. Add RIPA lysis buffer for lysis, place it on ice for 30 min, and extract total protein with a protein extraction reagent. After centrifuging at 13000 rpm for 10 min, take the lysate. Then, centrifuge the lysate again with a high-speed centrifuge and collect the supernatant, aliquot it, and store it in a -20°C refrigerator for later use. After measuring the BCA protein concentration of the samples, detect protein expression by Western Blotting experiment.

[0049] The results are as Figure 2 shown. The expression levels of IL-6, IL-17A, Tryptase, MMP13, and MPO in the OVA group of mice were all higher than those in the blank group. Both the PFH and 8M groups could down-regulate the expression of the above-mentioned inflammatory factors in OVA-induced allergic asthma mice, and the effect of 8M was stronger than that of PF.

[0050] The results are as Figure 4 shown. Different doses of 8-benzoylpaeoniflorin (8L, 8M, 8H) could all down-regulate the expression of the above-mentioned inflammatory factors IL-6, Tryptase, MMP13, and MPO in the lung tissue of OVA-induced allergic asthma mice to a certain extent, and showed a certain drug dose-dependence.

[0051] 1.7 Determination of the toxicity of 8-benzoylpaeoniflorin to BEAS-2B cells by MTT method

[0052] Accurately weigh 0.376 mg of 8-benzoylpaeoniflorin (standard product), add 100 μL of cell culture-grade DMSO solution and pipette to mix well to dissolve it, and prepare a mother liquor of 8-benzoylpaeoniflorin with a concentration of 10 mmol / L. When in use, pipette an appropriate amount of the mother liquor and add it to DMEM incomplete medium, and dilute it in gradients to obtain a series of concentrations of 200.00 μmol / L, 100.00 μmol / L, 50.00 μmol / L, 25.00 μmol / L, 12.50 μmol / L, 6.25 μmol / L, 3.125 μmol / L, and 1.5625 μmol / L. After filtering and sterilizing with a 0.22 μm filter head, it is ready for use.

[0053] Take BEAS-2B cells in the logarithmic growth phase, digest them with white trypsin, aspirate the supernatant after centrifugation, add the culture medium, and adjust the cell density to 8×10 4Inoculate 100 μL (at a density of cells / mL) into a 96-well plate and culture it in an incubator at 37 °C with 5% CO2 and saturated humidity. After overnight incubation, aspirate and discard the supernatant. Add 100 μL of 8-debenzoylpaeoniflorin solution at different concentrations to each well, and set up a blank control group with DMEM incomplete medium. Set up 6 replicates for each group. After 24 h of culture, add 100 μL of incomplete medium and 10 μL of MTT solution (5 mg / mL) to each well, and continue to culture for 4 h. Then, gently aspirate and discard the supernatant, add 150 μL of DMSO solution to each well, gently tap, and shake on a shaker for 5 - 10 min. Measure the absorbance at a wavelength of 490 nm using a multifunctional microplate reader.

[0054] The results are as Figure 5 shown. The toxicity of 8-debenzoylpaeoniflorin was evaluated by the MTT method, and it had no effect on the viability of BEAS-2B cells at concentrations below 25 μM.

[0055] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of 8-desbenzoylpaeoniflorin as the sole active ingredient in the preparation of a drug for treating asthma.

2. The application according to claim 1, wherein The asthma is allergic asthma.

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