KR20240003190A - Composition for treating vascular inflammation comprising Shinjulactone A - Google Patents

Abstract

The present invention relates to a composition for preventing or treating vascular inflammation containing Sinjulactone A as an active ingredient. The composition containing Sinjulactone A according to the present invention does not affect immune cells such as macrophages and endothelial cells. It effectively inhibits atherosclerosis regulated by NF-kB by binding in a specific manner, and can be used as a pharmaceutical or food composition useful for targeting tissue or improving, preventing or treating target-specific inflammation. .

Description

Composition for treating vascular inflammation comprising Shinjulactone A}

The present invention provides a composition for preventing or treating vascular inflammation containing sinjulactone A; And it relates to a food composition for improvement or prevention.

Cardiovascular disease is currently a leading cause of death worldwide. In particular, atherosclerosis is a disease that includes heart attacks and strokes, and accounts for most deaths due to cardiovascular disease. Atherosclerosis is a chronic inflammatory disease, and immune cell recruitment to the endothelium leads to atherosclerotic plaque development under hyperlipidemic conditions. Endothelial cells activate the key inflammatory mediator NFkB, which transcriptionally upregulates cell adhesion molecules including Vcam-1, Icam-1, E-selectin, and MCP-1 for endothelial recruitment of monocytes, such as impaired blood flow or multiple inflammatory ligands. Responds to inflammatory stimuli.

IL-1β is an important proinflammatory cytokine produced by macrophages and endothelial cells through the inflammatory pathway (Inflammasome pathway) and plays an important role in arteriosclerosis. Experimental results have also shown that antibody-mediated blockade of IL-1β reduces atherosclerosis-related cardiovascular events in patients. Clinical trials also showed a statistically significant increase in infection rates among patients treated with IL-1β blocking antibodies, suggesting adverse effects of systemic IL-1β blockade on immune function.

Endothelial to mesenchymal transition (EndMT) is a dedifferentiation process of endothelial cells that convert to mesenchymal cell types. During this process, endothelial cells lose several endothelial cell-specific marker proteins, such as VE-cadherin and PECAM, and begin to express mesenchymal a-SMA, vimentin, and fibronetin. There is accumulating evidence indicating that EndMT is involved in several vascular diseases, including fibrosis, PAH, and atherosclerosis. Mesenchymal cells induced by EndMT induce the secretion of pro-inflammatory molecules in atherosclerosis and affect the regulation of matrix and collagen production and plaque stabilization. EndMT induction in vitro requires TGF-β and inflammatory stimuli such as disrupted blood flow or IL-1β.

Endothelial inflammatory signaling plays an important role in atherosclerosis by inducing NFkB-dependent cell adhesion molecule (CAM) expression and monocyte recruitement. Conventional inhibitors, such as Bay 11-782, a well-known NFkB inhibitor, showed significant cytotoxicity in the long-term treatment of atherosclerosis.

Statins, currently widely used as a treatment for arteriosclerosis, were successful in treating or preventing arteriosclerosis by blocking LDL synthesis, but had serious side effects such as muscle pain and increased incidence of diabetes. Some preclinical studies and clinical trials have suggested that anti-inflammatory drugs may be an alternative treatment for atherosclerosis, but continued suppression of systemic inflammation may increase the risk of infection.

Therefore, there is an urgent need to develop drug candidates for the prevention or treatment of atherosclerosis that efficiently block IL-1β-induced NFkB activation and monocyte recruitment in endothelial cells, but do not affect NFkB activation induced by immunogenic substances in macrophages. It is happening.

The present inventors have made extensive research efforts to develop a drug that is effective in preventing or treating atherosclerosis without affecting endothelial cell survival rate. As a result, we completed the present invention by confirming that Sinjulactone A efficiently blocks IL-1β-induced NFkB activation and protein mobilization in endothelial cells while having little effect on LPS-induced NFkB activation in macrophages. It has been done.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating vascular inflammation containing Sinjulactone A as an active ingredient.

Another object of the present invention is to provide a food composition for improving or preventing vascular inflammation containing Sinjulactone A as an active ingredient.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention relates to a composition for preventing or treating vascular inflammation containing Shinjulactone A as an active ingredient.

In the present invention, Sinjulactone A is a quasinoid of Ailanthus altissima Swingle and is a substance used to treat colds and stomach diseases in Asian countries. Unlike the very similar quassinoid Ailanthone (which has a carbonyl group at C2 instead of a hydroxyl group), which has been widely studied for its anticancer effects, little is known about the function of Ailanthone A, except for its weak antitumor activity.

In the present invention, Shinju Lactone A may include a compound represented by the following formula (I), but is not limited thereto.

[Formula I]

In the present invention, Shinju Lactone A has a molecular weight of 378.4 and a molecular formula of C ₂₀ H ₂₆ O ₇ .

In the present invention, vascular inflammation may be caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia, but is limited thereto. That is not the case.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to achieve the efficacy or activity of Sinjulactone A.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, and silicic acid. Including, but not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. That is not the case. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

The pharmaceutical composition according to the present invention can be administered to mammals, including humans, through various routes. The administration method may be any commonly used method, for example, it may be administered orally, through the skin, intravenously, intramuscularly, subcutaneously, etc. For example, it may be administered orally.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, body weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

The composition of the present invention may further contain pharmaceutically suitable and physiologically acceptable auxiliaries such as carriers, excipients, and diluents in addition to the mixed extract.

Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulating, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, sucrose ( It can be prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Preparations for oral use include suspensions, oral solutions, emulsions, syrups, ointments, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, transdermal preparations, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

Preparations for suppositories include witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

In an embodiment of applying the composition of the present invention to humans, the composition of the present invention can be administered alone, but is generally mixed with a pharmaceutical carrier selected in consideration of the administration method and standard pharmaceutical practice (standard phamaceutical practice). may be administered.

For example, the compositions of the present invention may be administered in the form of tablets containing starch or lactose, in the form of capsules alone or with excipients, or in the form of elixirs or suspensions containing flavoring or coloring chemicals. It may be administered orally, intraorally, or sublingually. These liquid preparations may contain suspending agents (e.g. methylcellulose, semi-synthetic glycerides such as witepsol or a mixture of apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric It can be formulated with pharmaceutically acceptable excipients such as mixtures of glycerides).

The administered dose of the extract-containing composition of the present invention may vary depending on the patient's age, weight, gender, dosage form, health condition, and disease level, and may be administered once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. It may also be administered in divided doses.

In the present invention, the pharmaceutical composition may be administered with Sinjulactone A in the range of 1 to 10 mg/day, 1 to 8 mg, for example, 5 mg/day, per kg of body weight.

Another aspect of the present invention relates to a food composition for improving or preventing vascular inflammation containing Shinjulactone A as an active ingredient.

In the present invention, Shinju Lactone A may include a compound represented by the following formula (I), but is not limited thereto.

[Formula I]

In the present invention, Shinju Lactone A has a molecular weight of 378.4 and a molecular formula of C ₂₀ H ₂₆ O ₇ .

In the present invention, vascular inflammation may be caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia, but is limited thereto. It doesn't work.

When using the food composition of the present invention as a food additive, the food composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. In general, when manufacturing food or beverages, the food composition of the present invention may be added in an amount of 15% by weight or less, for example, 10% by weight or less, based on the raw materials.

The beverage may contain various flavors or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, and alcohol. , may contain carbonating agents used in carbonated drinks, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The proportions of these additives can also be appropriately selected by those skilled in the art.

In the food composition, content that overlaps with the pharmaceutical composition is omitted in consideration of the complexity of the present specification.

The present invention relates to a composition for preventing or treating vascular inflammation containing Sinjulactone A as an active ingredient. The composition containing Sinjulactone A according to the present invention does not affect immune cells such as macrophages and endothelial cells. Since it effectively inhibits atherosclerosis regulated by NF-kB by binding in a specific manner, it can be used as a pharmaceutical or food composition useful for targeting tissues or preventing or treating target-specific inflammation.

Figure 1 is a structural formula showing the chemical structure of Shinjulactone A according to an embodiment of the present invention.
Figure 2 is a diagram showing the results of immunoblotting of p-p65 and actin according to an embodiment of the present invention.
Figure 3 is a graph showing the quantitative results of immunoblotting of p-p65 and actin according to an embodiment of the present invention.
Figure 4 is a diagram showing monocyte adhesion to endothelial cells according to an embodiment of the present invention.
Figure 5 is a graph showing the results of counting monocyte adhesion to endothelial cells according to an embodiment of the present invention.
Figure 6 is a diagram showing the results of immunoblotting of p-p65 and actin after treatment with Sinjulactone A according to an embodiment of the present invention.
Figure 7 is a graph showing the quantitative results of immunoblotting of p-p65 and actin after treatment with Sinjulactone A according to an embodiment of the present invention.
Figure 8 is a graph showing the results of measuring cell viability after treatment with Sinjulactone A according to an embodiment of the present invention.
Figure 9 is a diagram showing the results of evaluating the levels of endothelial markers and mesenchymal markers by treatment with IL-1β and TGF-β1 after treatment with Sinjulactone A according to an embodiment of the present invention.
Figure 10 is a graph showing the quantitative results of immunoblotting after treatment with Sinjulactone A, IL-1β, and TGF-β1 according to an embodiment of the present invention.
Figure 11 is a diagram showing intercellular adhesion of BAEC cells on the 2nd and 5th days after treatment with Sinjulactone A and then with IL-1β and TGF-β1 according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Immunoblotting

1-1. Reagent preparation

The antibodies used for immunoblotting in this study were phospho-NFkB p65 (S ⁵³⁶ , Cell signaling technology), beta-actin (Cell signaling technology), anti-rabbit IgG, and anti-mouse IgG (R&D systems). . LPS (1ug/ml used), IL-1β (10ng/ml or 20ng/ml used), and TGF-b1 (10ng used) were purchased from Sigma. Shinjulactone A (Shinjulactone A) used in this experiment was separated and purified as a single component from low-white bark at the Korea Oriental Medicine Promotion Agency.

1-2. cell culture

Primary bovine aortic endothelial cells (BAEC) were isolated and maintained in DMEM (Dulbecco Modified Eagle Medium, GenDEPOT) supplemented with 10% FBS, penicillin, and streptomycin. All cell types were maintained at 37°C in a 5% CO ₂ incubator. THP-1 cells were maintained in RPMI-1640 supplemented with 10% FBS, penicillin, streptomycin, and 0.05 mM β-mercaptoethanol. Macrophage Raw 264.7 cells were cultured in DMEM containing 10% FBS and penicillin and streptomycin.

1-3. Endothelial to mesenchymal transition

One day after reapplication, cells were pretreated with sinjulactone A (10 uM) and DMSO for 1 hour as a control before incubation with TGF-b1 (10 ng/ml) and IL-1b (10 ng/ml). The medium was changed every two days to supply new ligand. After 2 and 5 days, cell morphology was examined and harvested for immunoblotting using EndMT marker antibody.

1-4. Immunoblotting

Equal amounts of protein samples were loaded into the wells of a 10%-polyacrylamide SDS-page gel, and the proteins were denatured with SDS-running buffer. Proteins were transferred to NC membrane (Nitrocellulose Filter, GE Healthcare Life science), blocked with 5% skim milk, and the membrane was incubated with p-p65 and actin antibodies overnight at 4°C. After washing with TBST three times for 10 minutes, anti-rabbit IgG and anti-mouse IgG secondary antibodies were incubated for 1 hour at room temperature. After washing with TBST three times for 10 minutes each, images were obtained using a chemiluminescent substrate (Thermofisher). Results were quantified using Image J and Prism5 software.

1-5. Monocyte adhesion

One day after cell seeding, sinjulactone A (1 uM, 10 uM, Figure 1) or DMSO as a control was added to endothelial cells (EC). After 1 hour, the cells were treated with IL-1β (20 ng/ml). After 6 hours, THP-1 cells were centrifuged and resuspended in 5% BSA (Bovine Serum Albumin, GeorgiaCHem) in HBSS (Hanks's Balanced Salt Solution, Gibco). After washing the cells once with PBS, the resuspended THP-1 cells were cultured on the endothelial cell monolayer for 30 minutes in an incubator with 5% CO ₂ at 37°C. After 30 minutes, unbound THP-1 cells were washed four times with 0.5% BSA HBSS. After fixing the cells with a 4% paraformaldehyde solution in PBS (USB) for 10 minutes, bound monocytes were counted under a microscope. Results were quantified using Prism 5 software.

1-6. cell viability

Bovine aortic endothelial cells were treated with Sinjulactone A (1 to 10 uM) or BAY 11-782 (1 to 10 uM) and maintained at 37°C for 5 days in a 5% CO ₂ incubator. Viable cells were counted every 24 hours after trypan blue staining (Thermofisher) using a countess II FL automatic cell counter, and the results were quantified using Prism 5 software.

Example 2. Evaluation of inhibition of vascular inflammation by Sinjulactone A

To investigate the effect of renal lactone A on vascular inflammation, NFkB activation was examined. NF-kB activation was monitored by immunoblotting for pS ⁵³⁶ -p65, which gradually decreased as the dose of sinjulactone A increased compared to the DMSO control, and the results are shown in Table 1 and Figures 2 and 3 below. .

Shinjulactone A Bay um 0 0.2 1.0 5.0 10.0 1.0 IL-1B (20ng) - + - + - + - + - + - + Mean One 3.50291 0.83343 1.82712 0.7456 1.68598 0.61719 1.04549 0.4273 0.51869 0.41948 0.88859 S.E. 0 1.96012 0.17113 0.79784 0.18058 0.6001 0.17132 0.11664 0.06226 0.06493 0.13752 0.19378

As can be seen in Figures 2 and 3, the NF-kB inhibitor Bay 11-782 almost completely blocked NF-kB activation at a concentration of 1 uM, and 5 uM sinjulactone A induced the same level of NFkB inhibition.

Example 3. Sinjulactone A-mediated monocyte attachment

Adhesion of monocytes to endothelial cells leads to migration of the endothelial cells and further inflammation. Monocyte THP-1 cells were added to the endothelial cells treated with Sinjulactone A, and incubated for 30 minutes. Then, after washing the unbound monocytes, the attached monocytes were counted and quantified, and the results are shown in Figures 4 and 5.

As can be seen in Figures 4 and 5, monocyte adhesion to IL-1β-induced bovine aortic endothelial cells was confirmed to be significantly reduced in cells treated with Sinjulactone A, supporting the anti-inflammatory activity of Sinjulactone A.

Example 4. Blocking LPS-induced NF-kB activation in macrophages

LPS is well known to induce the inflammasome pathway in macrophages by activating NF-kB. We tested whether synjulactone A-dependent NF-kB inhibition was specific to endothelial cells, and the results are shown in Figures 6 and 7.

As can be seen in Figures 6 and 7, Sinjulactone A, as a Bay compound, does not suppress inflammation in endothelial cells by directly regulating the NF-kB activation mechanism, but rather regulates cell type-specific or ligand-specific signaling. was able to confirm.

Example 5. Cytotoxicity evaluation of Sinjulactone A

The toxicity of cultured bovine aortic endothelial cells treated with Sinjulactone A (1 to 10 uM) was compared with control and BAY 11-782 (1 to 10 uM) treated cells. Cells were observed for 5 days after treatment. Surviving cells were counted every 24 hours using an automated cell counter after trypan blue staining, and the results are shown in Table 2 and Figure 8.

days DMSO Shinjulactone A BAY 1 uM S.E. 1 uM S.E. 10 uM S.E. 1 uM S.E. 10 uM S.E. One 85.24752 1.43275 88.25719 3.68963 91.96803 1.16663 84.80286 1.3052 58.47164 7.10787 2 85.35791 2.12067 87.34491 2.9429 87.75641 1.98796 85.91787 2.47553 42.48043 17.96584 3 89.77519 0.33776 89.19176 0.74297 86.9964 3.70314 84.25221 2.59972 48.21648 15.40378 4 79.91293 3.86046 81.92299 3.93849 86.53237 2.53093 85.28011 0.75721 59.52763 13.0193 5 85.36943 1.43605 86.00349 3.4075 82.57991 2.7129 84.72315 3.83799 43.77423 12.10106

As can be seen in Table 2 and Figure 8, the survival rate of cells treated with Sinjulactone A (1 to 10 uM) was 90% higher, similar to that of untreated cells, which means that Sinjulactone A (1 to 10 uM) 10 uM) indicates no toxicity to cells.

Example 6. Evaluation of reduction of endothelial-mesenchymal metastasis of Sinjulactone A

The effect of Sinjulactone A on endothelial-mesenchymal transition was investigated. Specifically, endothelial cells treated with 0 uM and 10 uM Sinjulactone A, respectively, were treated with IL-1β and TGF-β1, and the levels of endothelial and mesenchymal markers were evaluated, and the results are shown in Figures 9 to 11.

As can be seen in Figures 9 to 11, the down-regulation of VE-cadherein and the up-regulation of a-SMA were partially blocked 5 days after Sinjulactone A treatment. In the absence of IL-1β and TGF-β1, vascular endothelial cells had a cobblestone-like phenotype and were closely attached to each other. In addition, when co-treated with IL-1β and TGF-β1 for 2 days, endothelial cells showed a spindle-shaped shape and loss of cell-cell adhesion, but when treated with Sinjulactone A, the cell shape changed significantly compared to the untreated control group and the simultaneous treatment group. There was no. Additionally, in the samples after 5 days, Sinjulactone A still maintained intercellular adhesion of vascular endothelial cells.

Claims (6)

A pharmaceutical composition for preventing or treating vascular inflammation containing Shinjulactone A as an active ingredient. The pharmaceutical composition for preventing or treating vascular inflammation according to claim 1, wherein the Sinjulactone A includes a compound represented by the following formula (I).
[Formula I]
The method of claim 1, wherein the vascular inflammation is caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia. Pharmaceutical composition for preventing or treating vascular inflammation. A food composition for improving or preventing vascular inflammation containing Shinjulactone A as an active ingredient. The food composition for improving or preventing vascular inflammation according to claim 4, wherein the Sinjulactone A includes a compound represented by the following formula (I).
[Formula I]
The method of claim 1, wherein the vascular inflammation is caused by one selected from the group consisting of atherosclerosis, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, cerebrovascular disease, stroke, aneurysm, and arrhythmia. Food composition for improving or preventing vascular inflammation.