A composition for ischemic bowel disease and use thereof

Abstract

The application discloses a composition for ischemic enteropathy and application thereof, and relates to the technical field of medicine preparation. The composition comprises ferulic acid, ligustilide, angelica archangelica lactone A, gentiopicroside, swertiamarin, rhizomarhyanin and loganin acid. The composition for ischemic enteropathy can regulate FAK / VE-cadherin, MLCK / MLC and TGF-beta1 / SMAD3 pathways to improve vascular permeability, maintain intestinal barrier function and inhibit collagen deposition, so that the multiple components and multiple pathways jointly play the efficacy of treating ischemic enteropathy. The application solves the problem that there is no medicine for treating ischemic enteropathy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine preparation, in particular to a composition for ischemic enteropathy and application thereof. BACKGROUND

[0002] Ischemic enteropathy is a disease caused by insufficient blood supply to the intestinal tract, resulting in local intestinal tissue necrosis or even infarction. With the aggravation of population aging in recent years, the incidence and mortality of ischemic enteropathy are still rising year by year, which is an important problem to be solved in clinic.

[0003] Clinically, surgical, thrombolytic and interventional means are often used to restore blood supply as soon as possible, but this will lead to more serious reperfusion injury, increasing the difficulty of treatment of the disease. The etiology of ischemic enteropathy involves acute and chronic mesenteric ischemia, vascular obstructive and non-obstructive ischemia, and the pathological mechanism is complex, and there is no ideal treatment measure at present. Therefore, there is an urgent need for a drug for treating ischemic enteropathy. SUMMARY

[0004] In order to solve the above technical problems, the purpose of the present application is to provide a composition for ischemic enteropathy and application thereof, so as to solve the problem that there is no drug for treating ischemic enteropathy at present.

[0005] The technical scheme for solving the above technical problems of the present application is as follows: a composition for ischemic enteropathy is provided, which comprises ferulic acid, ligustilide, ostruthin A, gentiopicroside, swertiamarin, rhizomaroside and loganin acid.

[0006] The composition of the present application is derived from traditional Chinese medicine Gentiala and Chuanxiong, and the curative effect of the composition of the present application is significantly higher than that of a single compound. The composition can protect vascular endothelial cells to reduce vascular permeability, reduce intestinal epithelial damage to maintain intestinal barrier function, and inhibit abnormal proliferation of fibroblasts to reduce intestinal adhesion, thereby showing the pharmacodynamic effect of treating ischemic enteropathy.

[0007] On the basis of the above technical scheme, the present application can be further improved as follows:

[0008] Further, the molar ratio of ferulic acid, ligustilide, ostruthin A, gentiopicroside, swertiamarin, rhizomaroside and loganin acid is 0.1-5:0.1-5:0.1-20:0.1-5:0.1-5:0.1-5:0.1-5.

[0009] Further, the molar ratio of ferulic acid, ligustilide, ostruthin A, gentiopicroside, swertiamarin, rhizomaroside and loganin acid is 5:1:1:5:1:1:5.

[0010] Further, the molar ratio of ferulic acid, ligusticum chalcone, angelica archangelica lactone A, gentiopicroside, swertiamarin, rhizoma dryopteris lactone and loganin acid is 1:1:1:1:1:1:1.

[0011] The present application also provides the use of the above-mentioned composition in the preparation of a medicament for ischemic enteropathy.

[0012] Further, the ischemic enteropathy includes acute mesenteric ischemia, chronic mesenteric ischemia and ischemic colitis.

[0013] The present application also provides a medicament for ischemic enteropathy, comprising the above-mentioned composition for ischemic enteropathy.

[0014] Further, the dosage form of the medicament is an injection, a powder, a pill, a tablet, a microcapsule, a soft capsule, a film, a paste, a tincture, a granule, an aerosol permitted in pharmacy.

[0015] The present application has the following beneficial effects:

[0016] 1. The composition of the present application can improve intestinal mucosal atrophy, intestinal villus shedding, reduce inflammatory infiltration of intestinal epithelial cells, and maintain intestinal barrier function through the MLCK / MLC pathway based on intestinal epithelial cells.

[0017] 2. The composition of the present application can reduce vascular endothelial cell damage and improve intestinal vascular permeability through the FAK / VE-cadherin pathway based on vascular endothelial cells.

[0018] 3. The composition of the present application can reduce intestinal adhesion and inhibit collagen distribution and deposition in adhesion tissue through the TGF-β1 / SMAD3 pathway based on fibroblasts.

[0019] 4. The composition for ischemic enteropathy of the present application can improve vascular permeability, maintain intestinal barrier function and inhibit collagen deposition by regulating the FAK / VE-cadherin, MLCK / MLC and TGF-β1 / SMAD3 pathways, and further multiple components and multiple pathways jointly play the effect of treating ischemic enteropathy.

[0020] 5. The composition of the present application can be used in different proportions, and formulated into an injection, or a powder, or a pill, or a tablet, or a microcapsule, or a soft capsule, or a film, or a paste, or a tincture, or a granule, or an aerosol used in clinic, and multiple components and multiple targets jointly play the effect of treating ischemic enteropathy. DETAILED DESCRIPTION

[0021] The following description is provided so as to enable those skilled in the art to readily understand the principles and features of the present application. The description is merely illustrative of the present application and should not be construed as limiting the scope of the present application. Unless specifically noted, the conditions, measures or materials described in the examples are conventional conditions, measures or materials used by those skilled in the art. Unless specifically noted, the reagents or instruments used are conventional products available on the market.

[0022] Example 1

[0023] A composition for ischemic enteropathy comprising ferulic acid, ligustilide, ostruthin, gentiopicroside, swertiamarin, rhizomarabin and loganin acid (molar ratio of 5:1:1:5:1:1:5).

[0024] A medicament for ischemic enteropathy comprising the above-mentioned composition for ischemic enteropathy.

[0025] Example 2

[0026] A composition for ischemic enteropathy comprising ferulic acid, ligustilide, ostruthin, gentiopicroside, swertiamarin, rhizomarabin and loganin acid (molar ratio of 0.5:0.1:0.1:0.5:0.1:0.1:0.5).

[0027] A medicament for ischemic enteropathy comprising the above-mentioned composition for ischemic enteropathy.

[0028] Example 3

[0029] A composition for ischemic enteropathy comprising ferulic acid, ligustilide, ostruthin, gentiopicroside, swertiamarin, rhizomarabin and loganin acid (molar ratio of 1:1:1:1:1:1:1).

[0030] A medicament for ischemic enteropathy comprising the above-mentioned composition for ischemic enteropathy.

[0031] Example 4

[0032] A composition for ischemic enteropathy comprising ferulic acid, ligustilide, ostruthin, gentiopicroside, swertiamarin, rhizomarabin and loganin acid (molar ratio of 5:5:20:5:5:5:5).

[0033] A medicament for ischemic enteropathy comprising the above-mentioned composition for ischemic enteropathy.

[0034] Comparative Example 1

[0035] A composition for ischemic enteropathy comprising ferulic acid, gentiopicroside and loganin acid (molar ratio of 5:5:5).

[0036] A medicine for ischemic enteropathy, comprising the composition for ischemic enteropathy described above.

[0037] Test Example

[0038] I. Evaluation of intestinal mucosal injury

[0039] The composition prepared in Example 1 and Comparative Example 1 was subjected to an evaluation experiment of intestinal mucosal injury by the gavage method. The method of implementation was as follows: C57 BL / 6J mice were used, and an intestinal ischemia-reperfusion injury (IIRI) model was used to simulate ischemic enteropathy, i.e., after clamping the superior mesenteric artery for ischemia for 45 min and reperfusion for 90 min, the mice were divided into a normal group, a model group, an Example 1 group, and a Comparative Example 1 group. After the modeling was completed, the mice were continuously administered by gavage for 7 days, and then were sacrificed to obtain samples, followed by H&E staining, evaluation of the injury of intestinal tissue by Chiu's score, and observation of intestinal adhesion. The results are shown in Table 1.

[0040] Table 1 Effects on intestinal mucosa

[0041]

[0042] Note: *p < 0.05 vs. the Example 1 group.

[0043] As shown in Table 1, after modeling, the intestinal villi were damaged, a large number of inflammatory cells infiltrated the crypt, and intestinal adhesion was generated; the composition of Example 1 could significantly reduce intestinal mucosal injury and improve adhesion generation, and reduce the Chiu's score, and the therapeutic effect was significantly different from that of the composition of Comparative Example 1 containing three components. Therefore, the composition of the present application has irreplaceability, synergistic effect between the components of the composition, and better efficacy than a single component, and has a good effect of treating ischemic enteropathy

[0044] II. Dose evaluation

[0045] 1. The composition prepared in Example 2 was used for intestinal mucosa injury evaluation experiment by gavage. The implementation method was as follows: C57 BL / 6J mice were taken, and ischemia-reperfusion injury (IIRI) model was used to simulate ischemic enteropathy, that is, the superior mesenteric artery was clamped for 45 min and then reperfused for 90 min. The mice were divided into normal group, model group, composition low-dose group (ZHWL), composition medium-dose group (ZHWM) and composition high-dose group (ZHWH). The mouse dosages of ferulic acid, ligustilide, ostrutholide A, gentiopicroside, swertiamarin, rhemaein and loganin acid in ZHWL group were 0.5 μmol / kg, 0.1 μmol / kg, 0.1 μmol / kg, 0.5 μmol / kg, 0.1 μmol / kg, 0.1 μmol / kg and 0.5 μmol / kg, respectively; the mouse dosages of ferulic acid, ligustilide, ostrutholide A, gentiopicroside, swertiamarin, rhemaein and loganin acid in ZHWM group were 1 μmol / kg, 0.2 μmol / kg, 0.2 μmol / kg, 1 μmol / kg, 0.2 μmol / kg, 0.2 μmol / kg and 1 μmol / kg, respectively; the mouse dosages of ferulic acid, ligustilide, ostrutholide A, gentiopicroside, swertiamarin, rhemaein and loganin acid in ZHWH group were 2 μmol / kg, 0.4 μmol / kg, 0.4 μmol / kg, 2 μmol / kg, 0.4 μmol / kg, 0.4 μmol / kg and 2 μmol / kg, respectively. After the modeling was completed, the mice were continuously gavaged for 7 days, then were sacrificed and the samples were taken, followed by H&E staining, and Chiu's score was used to evaluate the injury of intestinal tissue. The results are shown in Table 2.

[0046] Table 2 Effects of different doses of composition on intestinal mucosa

[0047]

[0048] Note: *p<0.05 vs. IIRI group, **p<0.01 vs. IIRI group; # p<0.05 vs. ZHWH group, ## p<0.01 vs. ZHWH group.

[0049] As shown in Table 2, after the mice in the model group were continuously gavaged for 7 days, the H&E staining results showed that the atrophy of intestinal mucosa, the shedding of intestinal villi and the inflammatory infiltration of intestinal epithelial cells were significantly reduced after the composition treatment, and the Chiu's score of intestinal tissue injury was reduced.

[0050] 2. Serum samples were collected from mice in each group, and the levels of D-dimer (D2D) and intestinal fat-binding protein (iFABP) were measured strictly according to the ELISA kit instructions. D2D is closely related to thrombus formation in the bloodstream, and iFABP can reflect the severity of villous damage during intestinal ischemia. Both of these indicators are biomarkers of acute mesenteric ischemia. GraphPad Prism 8.4.0 software was used to analyze and statistically analyze the data, and the results are shown in Table 3.

[0051] Table 3 Effects of the composition on serum D2D and iFABP levels in mice

[0052]

[0053] Note: *p<0.05 vs. model group.

[0054] As shown in Table 3, the levels of D2D and iFABP were significantly increased in the model group, while the composition of the present invention can significantly reduce the levels of D2D and iFABP, thus alleviating acute mesenteric ischemia injury.

[0055] III. Effects on the expression of FAK / VE-cadherin pathway proteins

[0056] The effect of the composition prepared in Example 1 on the expression of FAK (focal adhesion kinase) / VE-cadherin (cadherin) pathway proteins was detected. The cells used in the experiment were human umbilical vein endothelial cells (HUVEC) (ATCC Cell Bank, USA), and the FAK phosphorylation inhibitor Y15 (10 μmol / L) (Shanghai Yuanye Biotechnology Co., Ltd.) was used as a control. The conventional Western blot method was used for testing.

[0057] The specific detection method was as follows: First, an OGD / R model was established, in which HUVEC cells were placed in a carbon dioxide incubator (containing 5% CO2, 37℃) and cultured in DMEM / F-12 1:1 medium containing 20% ​​fetal bovine serum. The medium was changed every 2 days, and cells in the logarithmic growth phase were used for experiments. A three-gas incubator and oxygen-glucose deprivation were used to simulate an ischemic environment. The modeling conditions were: hypoxia for 1 h, reoxygenation for 4 h. The results are shown in Table 4.

[0058] Table 4. Effects of the composition on FAK / VE-cadherin pathway protein expression.

[0059]

[0060]

[0061] Note: *p<0.05 vs. OGD / R group.

[0062] From Table 4, after OGD / R, the level of p-FAK was significantly increased, which was 4.44 times of the normal group, while the composition group, Y15 group and composition + Y15 group were 3.23, 1.64 and 1.83 times of the normal group respectively, indicating that the composition can significantly reduce the level of p-FAK; after OGD / R, the expression of VE-cadherin between cells was significantly reduced, which was 0.46 times of the normal group, and the level of p-β-Catenin was significantly increased, which was 2.90 times of the normal group, while the VE-cadherin of the composition group, Y15 group and composition + Y15 group was 0.66, 0.76 and 0.70 times of the normal group respectively, and the level of p-β-Catenin was 2.29, 1.68 and 1.57 times of the normal group respectively; indicating that the composition can significantly increase the expression of VE-cadherin and reduce the level of p-β-Catenin.

[0063] IV. Effect on the expression of MLCK / MLC pathway proteins

[0064] The composition prepared in Example 1 was detected for the effect on the expression of MLCK (myosin light chain kinase) / MLC (myosin light chain) pathway proteins, and the cells used in the test were normal human intestinal epithelial cells HIEC-6 (American ATCC cell library), and MLCK inhibitor ML-7 (Shanghai Yuan Ye Biological Technology Co., Ltd.) (10 μmol / L) was used as a control, and the conventional Western blot method was used for testing.

[0065] The detection method is as follows: first, an OGD / R model is established, that is, HIEC-6 cells are placed in a carbon dioxide incubator (containing 5% CO2, 37°C), and are cultured in DMEM / F-12 1:1 medium containing 20% fetal bovine serum, and the medium is changed every 2 days, and the cells in the logarithmic growth phase are used for the experiment. The ischemic environment is simulated by a three-gas incubator and oxygen-glucose deprivation. The modeling conditions are as follows: 1 h of hypoxia and 4 h of reoxygenation. The results are shown in Table 5.

[0066] Table 5 Effect of the composition on the expression of MLCK / MLC pathway proteins

[0067]

[0068]

[0069] Note: *p<0.05 vs. OGD / R group.

[0070] From Table 5, it can be seen that after OGD / R, the expression of MLCK increased, which was 2.77 times that of the normal group, while the expression of the composition group, the ML-7 group and the composition + ML-7 group was 2.32, 1.74 and 1.83 times that of the normal group, respectively, indicating that the composition of the application can reduce the expression of MLCK; after OGD / R, the phosphorylation level of MLC increased significantly, and the expression of tight junction proteins ZO-1 and Occludin decreased significantly, which was 5.34, 0.29 and 0.46 times that of the normal group, respectively, while the composition of the application can reduce the expression of MLCK, reduce the level of p-MLC and thus increase the expression of ZO-1 and Occludin, thereby protecting HIEC-6 intestinal epithelial cells.

[0071] V. Effect on TGF-β / SMAD3 Pathway Protein Expression

[0072] The composition prepared in Example 1 was detected for its effect on TGF-β (transforming growth factor-β) / SMAD3 (Smad homolog 3) pathway protein expression, and fibroblast L929 (American ATCC cell library) was used as the test cell, and TGF-β / SMAD3 pathway inhibitor SB431542 (10 μmol / L) (Shanghai Yuan Ye Biological Technology Co., Ltd.) was used as the control, and the conventional Western blot method was used for testing.

[0073] The implementation method is as follows: first, a TGF-β induced L929 proliferation and transdifferentiation model is established, and the L929 cells are placed in a carbon dioxide incubator (containing 5% CO2, 37℃), and are cultured in DMEM / F-12 1:1 medium containing 20% fetal bovine serum, and the medium is changed every 2 days, and the cells in the logarithmic growth phase are used for experiments. The modeling condition is that the L929 cells are treated with 2.5 ng / mL TGF-β1 for 72 h. The results are shown in Table 6.

[0074] Table 6 Effect of the composition on TGF-β / SMAD3 pathway protein expression

[0075]

[0076]

[0077] Note: *p<0.05 vs. TGF-β1 group.

[0078] From Table 6, after TGF-β1 stimulation, the level of p-SMAD3 increased by 3.34 times, the expression of Collagen I and α-SMA increased significantly, which were 1.93 and 1.64 times of the normal group; the level of p-SMAD3 of the composition group, the SB431542 group and the composition + SB431542 group was 1.63, 1.59 and 1.39 times of the normal group, the expression of Collagen I was 1.50, 1.28 and 1.22 times of the normal group, and the expression of α-SMA was 0.89, 1.04 and 1.10 times of the normal group. It is shown that the composition of the application can reduce the expression of Collagen I and α-SMA by reducing the level of p-SMAD3, and significantly inhibit the abnormal proliferation and differentiation of L929 cells.

[0079] The above description is merely preferred embodiments of the present application, but not to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composition for ischemic bowel disease, characterized in that, Including ferulic acid, ligustilide, angelicin A, gentiopicrin, swertiamarin, strychnine, and loganic acid; The molar ratio of ferulic acid, ligustilide, angelica lactone A, gentiopicrin, swertiamarin, strychnine, and loganic acid is 0.1-5:0.1-5:0.1-20:0.1-5:0.1-5:0.1-5:0.1-5:0.1-5.

2. The composition for ischemic bowel disease according to claim 1, characterized in that, The molar ratio of ferulic acid, ligustilide, angelica lactone A, gentiopicrin, swertiamarin, strychnine, and loganic acid is 5:1:1:5:1:1:

5.

3. The composition for ischemic bowel disease according to claim 1, characterized in that, The molar ratio of ferulic acid, ligustilide, angelica lactone A, gentiopicrin, swertiamarin, strychnine, and loganic acid is 1:1:1:1:1:1:1:

1.

4. Use of the composition for ischemic bowel disease according to any one of claims 1-3 in the preparation of a medicament for ischemic bowel disease.

5. A drug for ischemic bowel disease, characterized in that, The composition for ischemic bowel disease as described in any one of claims 1-3.

6. The medicament for ischemic bowel disease according to claim 5, characterized in that, The dosage form of the drug is pharmaceutically permissible as injection, powder, pill, tablet, microcapsule, soft capsule, film, ointment, tincture, granule, or aerosol.

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