KR20210055383A - Pharmaceutical composition for preventing or treating vascular diseases comprising securinega suffruticosa extract - Google Patents

Abstract

The present invention relates to a composition for preventing or treating vascular diseases, containing a Lepisorus thunbergianus extract. The present invention is particularly useful for preventing or treating hyperlipidemia. The Lepisorus thunbergianus extract of the present invention can be usefully used as a pharmaceutical composition, a food composition, or an animal feed composition for preventing, treating, or alleviating vascular diseases.

Description

A pharmaceutical composition for the prevention or treatment of vascular diseases comprising an extract of Japanese peppercorns {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING VASCULAR DISEASES COMPRISING SECURINEGA SUFFRUTICOSA EXTRACT}

The present invention relates to a composition for preventing or treating vascular diseases, particularly hyperlipidemia, comprising an extract of Securinega suffruticosa as an active ingredient.

The soft leaves in the early spring are edible as herbs, and the leaves and stem roots are used for medicinal purposes, and the name of the herbal medicine is Ilyeopchu (一葉萩). Ilyeopchu is spicy, bitter, and warm, and its medicinal effect strengthens blood vessels to help blood circulation, help spleen and kidneys, low back pain due to rheumatism, quadriplegia, hemiplegia, facial nerve palsy. , To treat sequelae such as polio.

Vascular diseases are caused by death and inflammation of vascular endothelial cells caused by inflammatory cytokines such as tumor necrosis factor-α (TNF-α). In particular, TNF-α is known to increase the activity of NF-κB, a representative inflammatory transcription factor, and through this, various inflammatory conjugation proteins, specifically vascular cell adhesion molecule-1 (VCAM-1), and intercellular It increases the expression of cell adhesion molecules (CAMs) such as intercellular adhesion molecules (ICAM-1) and E-selectin (endothelial cell selectin), which induces adhesion of immune cells to vascular endothelial cells. Increasing the inflammatory reaction.

In atherosclerosis, as a result of cholesterol deposits on the lining of blood vessels, an'atheroma' is formed, and the inside of the atheroma becomes thin like a porridge, and the surrounding area is surrounded by a hard fibrous membrane'sclerotic plaque', where bleeding occurs inside the atheroma. It means a disease in which a blood clot occurs in a blood vessel due to a rupture of the sclerotic plate or a blood clot occurs due to the fall of endothelial cells, the inner diameter of the blood vessel sharply narrows or the blood vessel is completely blocked, as a result of which the blood circulation to the periphery is impaired. . Atherosclerosis often occurs as a vascular complication of diseases such as obesity, diabetes, hypertension, and hyperlipidemia. In particular, when the concentration of LDL (low-density lipoprotein) is high in blood vessels, hyperlipidemia is caused. When LDL (low-density lipoprotein) adheres to the damaged blood vessel wall and is supplied to the tissue, it is oxidized by NO (nitric oxide) secreted by endothelial cells and macrophages. Oxidized LDL (Ox-LDL) stimulates endothelial cells, and increased oxidative stress on the surface of endothelial cells activates NF-κB, which plays an important role in the inflammatory response of vascular endothelial cells and vascular smooth muscle cells, through which interleukin Expression of CAMs such as VCAM-1, ICAM-1, and E-selectin by inflammatory cytokines such as -1(IL-1) and TNF-α, and monocyte chemoattractant protein (MCP-1), interleukin Increases the expression of -8 (IL-8) and the like. Through this process, monocytes in the blood pass through the endothelial cells, gather inside, and differentiate into macrophages, resulting in a vicious cycle of oxidizing LDL.

In addition, the process of macrophages predating LDL occurs.When the macrophages die, a lipid nucleus consisting mainly of the remains of macrophages, that is, an atheroma is formed, and the fibrous membrane surrounding the scleroderma is ruptured, resulting in a blood clot. Sclerosis develops. Therefore, the inflammatory response of monocytes and vascular endothelial cells following damage to vascular endothelial cells is an important pathophysiology of early atherosclerosis.

Existing anti-inflammatory drugs are often accompanied by various side effects, so the development of anti-inflammatory drugs with strong effects and relatively few side effects is constantly required. Therefore, the development of natural drugs with fewer side effects than synthetic drugs can be of great help to patients.

Meanwhile, it has been known that the extract of Securinega suffruticosa exhibits an inhibitory effect on osteoporosis and the like (Korean Patent Laid-Open No. 10-2013-0059960). However, until now, it has not been disclosed that the extract of the Japanese leaf chives exerts an effect on vascular diseases.

Under this background, the present inventors have attempted to develop a therapeutic agent useful for vascular diseases using natural products traditionally used as herbal or folk remedies in Korea, and the effect of monoleaflet extract on the expression of cell adhesion factor genes and proteins in vascular endothelial cells. And completed the present invention.

Korean Patent Application Publication No. 10-2013-0059960

An object of the present invention is to provide a pharmaceutical composition, food composition, or animal feed composition having few side effects and excellent in preventing or treating vascular disease.

Particularly, the present invention relates to the antihyperlipidemia effect of the extract of Securinega suffruticosa , and an antihyperlipidemia composition comprising an ethanol extract of one leaf chives capable of blocking the initial occurrence of hyperlipidemia as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention or treatment of vascular disease comprising the extract as an active ingredient.

The vascular disease may be selected from the group consisting of atherosclerosis, coronary artery disease, myocardial infarction, angina, hemangioma, stroke, cerebral infarction, arrhythmia, hypertension, hyperlipidemia, and heart failure, but is not limited thereto. Preferably, it is hyperlipidemia or atherosclerosis.

One leaf extract of the present invention can prevent or treat vascular disease by inhibiting the expression of an inflammatory protein selected from the group consisting of VCAM-1, ICAM-1, and IL-6.

One leaf extract of the present invention can exhibit a preventive or therapeutic effect on hyperlipidemia by inhibiting the adhesion of monocytes induced by oxidized low specific gravity protein (ox-LDL) in the blood.

One leaf extract of the present invention may be extracted using C1 to C6 alcohol, water, or a mixture thereof. It can preferably be extracted using ethanol, particularly preferably 80% ethanol.

The present invention provides a food composition for the prevention or improvement of vascular disease, comprising the extract as an active ingredient. The food composition may be a health functional food, a dairy product, a fermented product, or a food additive.

The present invention provides an animal feed composition for the prevention or improvement of vascular disease, comprising an extract of Japanese leaf chives as an active ingredient.

One leaf extract of the present invention has the effect of significantly inhibiting the expression of VCAM-1, ICAM-1, E-selectin or IL-6. In addition, the extract of the present invention exhibits an effect of preventing cell adhesion of mononuclear cells when administered with an oxidized low specific gravity lipoprotein (ox-LDL) that can cause hyperlipidemia to vascular endothelial cells, thereby exhibiting the effect of antihyperlipidemia.

Therefore, the extract of the present invention improves both the vascular inflammatory response due to cytokines and the vascular inflammatory response due to oxidized low specific gravity lipoproteins in vascular endothelial cells, thereby preventing, treating, and preventing vascular diseases, particularly hyperlipidemia or atherosclerosis, Or it has an improvement effect, and as a natural product-derived ingredient, there are few side effects, and there is a very low possibility of resistance even when used for a long period of time. As a result, the extract of the Japanese leaves of the present invention may be usefully used as a pharmaceutical composition, a food composition, or an animal feed composition for preventing, treating, or improving vascular disease.

1 is a graph showing cell viability when human vascular endothelial cells are treated with 80% ethanol extract of one leaf.
Figure 2 is a graph showing the results of the test confirming the inhibitory effect of the extract of Japanese leaves on the expression of VCAM-1 induced by TNF-α according to Example 3-1.
Figure 3 is a graph showing the test results confirming the inhibitory effect of the extract of the Japanese leaves on the expression of VCAM-1, ICAM-1, and IL-6 induced by TNF-α according to Example 3-2.
4 and 5 are graphs showing that the adhesion of HL-60 cells (human leukemia cells) induced by Ox-LDL in vascular endothelial cells is inhibited when the ethanol extract is treated by concentration.

Hereinafter, embodiments and examples of the present disclosure will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present disclosure. However, the present application may be implemented in various forms and is not limited to the embodiments and examples described herein.

In the entire specification of the present application, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise specified.

The present invention relates to a pharmaceutical composition for the prevention or treatment of vascular disease, comprising an extract of Japanese peppercorns as an active ingredient.

The term "extract" as used herein may mean a liquid component obtained by extraction for a certain period of time after adding a solvent to a target material, and a resultant product such as a solid component obtained by removing the solvent from the liquid component. In addition, in addition to the resulting product, it can be comprehensively interpreted as including all of the diluted solution of the resultant, the concentrated solution thereof, the preparation thereof, and the purified product.

In one embodiment, the monoleaflet extract of the present invention prevents or treats vascular disease by inhibiting the expression of an inflammatory protein selected from the group consisting of VCAM-1, ICAM-1, E-selectin, and IL-6.

As used herein, the term "cell adhesion molecules (CAMs)" refers to a subset of cell adhesion proteins involved in cell adhesion that exist on the surface of a cell and bind to other cells or extracellular matrix. Cell adhesion molecules include VCAM-1, ICAM-1, and E-selectin.

The term "vascular cell adhesion molecule-1 (VCAM-1)" as used herein is also referred to as a vascular cell adhesion molecule, and only after endothelial cells are stimulated by cytokines such as TNF-α, IL-1, IL-4, etc. It is expressed, and the expression lasts for 24 hours and refers to a type of cell adhesion molecule that mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to the vascular endothelium.

As used herein, the term "ICAM-1 (intercellular adhesion molecules)" is also referred to as an intercellular adhesion molecule, and when it is stimulated by cytokines such as IL-1 and TNF, its expression rapidly increases in vascular endothelium, macrophages, and lymphocytes. , It refers to a transmembrane protein that mediates white blood cells in the blood to enter the space under the endothelial cells after adhering to the blood vessel wall.

The term "E-selectin (endothelial cell selectin)" as used herein is expressed only in endothelial cells activated by cytokines such as IL-1 and TNF-α in inflamed tissues, and induces leukocytes to move to the injury site. It refers to a kind of cell adhesion protein that plays a role. Specifically, as E-selection weakly binds to white blood cells, temporary interactions are created and disappeared, white blood cells move around the endothelium of blood vessels. They are strongly bound to the surface of endothelial cells by chemokines secreted by damaged tissues and into the space under the endothelial cells. Let's go in.

In one embodiment, the vascular disease may be selected from the group consisting of atherosclerosis, coronary artery disease, myocardial infarction, angina, hemangioma, stroke, cerebral infarction, arrhythmia, high blood pressure, hyperlipidemia, and heart failure. Preferably, it may be hyperlipidemia or atherosclerosis.

One leaf extract of the present invention can exhibit a preventive or therapeutic effect on hyperlipidemia by inhibiting the adhesion of monocytes induced by oxidized low specific gravity protein (ox-LDL) in the blood.

A pharmaceutical composition comprising the extract of the present invention as an active ingredient may be formulated.

In one embodiment, the formulation of the pharmaceutical composition may be selected from the group consisting of powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, and injections.

One leaf extract of the present invention may be extracted using C1 to C6 alcohol, water, or a mixture thereof. It can be extracted using preferably ethanol, more preferably 80% ethanol.

The extraction method used in the present invention may use any method commonly used, for example, immersion (cold immersion or warm immersion), hot water extraction, ultrasonic extraction, or reflux cooling extraction method, but is not limited thereto.

The present invention provides a food composition for the prevention or improvement of vascular disease, comprising the extract as an active ingredient.

In one embodiment, the food composition may be a health functional food, a dairy product, a fermented product or a food additive.

In one embodiment, the food composition is a variety of nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and Salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may further be included.

The present invention provides an animal feed composition for the prevention or improvement of vascular disease, comprising an extract of Japanese leaf chives as an active ingredient.

The present invention is to be described in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Production Example 1]

Preparation of 80% ethanol extract of Japanese leaves

400g of leaves of Cheongsamsari (one leaf chives) purchased in Samcheok, Gangwon-do, and 4L of 80% ethanol were placed in an Erlenmeyer flask and left at room temperature for a week. The cooler was connected to the Erlenmeyer flask, heated at 80-90° C. for 2 hours, filtered with gauze, and centrifuged at 3,000 rpm for 20 minutes. It was concentrated in a rotary vacuum concentrator at 52℃ and speed 3~4 until the final volume reached 400 ml. Concentrated leaves were freeze-dried for 7 days to obtain powdery form, yielding 11.753%.

The 80% ethanol extract prepared in this way was used in the following examples.

In addition, methanol, propanol, butanol, and an aqueous solution thereof were added in the same manner as described above to prepare an extract.

[Example 1]

Cell culture

Human vascular endothelial cells (primary human umbilical vascular vein endothelial cells, HUVECs) were purchased from PromoCell. Cell culture is 0.02 ml/ml Fetal Calf Serum, 0.004 ml/ml Endothelial Cell Growth Supplement, 0.1 ng/ml Epidermal Growth Factor (recombinant human), 1 ng/ml Basic Fibroblast Growth Factor (recombinant human), 90 μg/ml Heparin , 1 μg/ml Hydrocortisone was cultured at 37°C in a 5% carbon dioxide incubator in Endothelial Cell Growth Medium. The culture medium was changed at intervals of 2-3 days, and when cells grew more than 80% of the culture dish area, they were passaged with 0.04%/0.03% trypsin-EDTA (PromoCell, Heidelberg, GER), and were not passaged more than 7 times. .

[Example 2]

Cytotoxicity test of Japanese leaf extract

In this example, the viability of human vascular endothelial cells was measured to evaluate the cytotoxicity of the extract of Ichidaceae. The survival rate of human vascular endothelial cells was analyzed using MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide).

One leaf extract 80% ethanol (IYC) was treated to human vascular endothelial cells by concentration, and cultured for 24 hours. After that, 10 μl of MTT solution (50 mg/ml) was added to the upper medium, and the plate was reacted at 37° C. for 3 hours. After removing the supernatant, 100 µl of DMSO was added and mixed by shaking at room temperature for 10 minutes, and the absorbance was measured at 540 nm with an ELISA reader at the same time without error over time. Each condition was tripled, and the experiment was repeated 5 times.

As a result, as shown in Figure 1, when 1 μg/ml to 50 μg/ml of ethanol extract (IYC) was treated, the cell viability was maintained the same compared to that of the normal group (untreated group). From this, it was confirmed that the concentration of 1 μg/ml to 50 μg/ml of the monoleaflet extract of the present invention does not show cytotoxicity, so that it can be used safely.

[Example 3]

Evaluation of inhibition of expression of cell adhesion molecules

In this example, the degree of suppression of the expression of cell adhesion molecules induced by cytokines was evaluated by the extract of Japanese chives. After pretreatment of the Japanese leaf extract by concentration, a protein expression test of the cell adhesion molecule by TNF-α stimulation was performed as follows.

Example 3-1: VCAM-1 expression inhibition measurement-Cell ELISA analysis

The expression level of VCAM-1 on the surface of vascular endothelial cells cultured in Example 1 was measured using cell ELISA.

^{After culturing by dispensing 1×10 4} cells/plate of endothelial cells in a 96 well plate, 80% ethanol extract (IYC) was added to each concentration after cultivation. After 6 hours, the culture supernatant was transferred to a new plate and incubated for 2 hours. After suctioning the culture supernatant, the detection antibody was treated for 2 hours and then suctioned. After washing, streptavidin-HRP was treated and incubated for 20 minutes, the substrate buffer was treated, and the stop solution was treated after 20 minutes to measure absorbance at 450 nm.

As a result, as shown in FIG. 2, when induced with TNF-α, VCAM-1 increased by about 2.7 times compared to the control group. On the other hand, in the case of pretreatment of 10 μg/ml, 20 μg/ml, and 50 μg/ml of monoleaf ethanol extract (IYC), VCAM-1 expression is concentration-dependent at about 2.0 times, about 1.7 times, and about 1.5 times for each concentration. Decreased to.

Example 3-2: VCAM-1, ICAM-1, IL-6 expression inhibition measurement-Western blot analysis

In the vascular endothelial cells cultured in Example 1, the expression levels of VCAM-1, ICAM-1, and IL-6 were measured using Western blot. 80% ethanol extract (IYC) was added for each concentration and incubated for 30 minutes, then treated at a concentration of TNF-α 50 ng/ml, and the cells were collected by scraping with a cell scraper after 6 hours. The collected cells were separated by cell homogenates (30 μg of protein) by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred on a nitrocellulose membrane. Membrane was washed three times with TBS-T [10mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween-20] for 5 minutes each, followed by a blocking process with 5% BSA for 1 hour. Then, monoclonal anti-mouse primary antibodies (VCAM-1, ICAM-1, IL-6) were incubated for 1 hour, and washed 3 times for 10 minutes each with TBS-T. It was treated with secondary antibody (goat anti-mouse-IgG conjugated to horseradish peroxidase) for 1 hour and washed 3 times with TBS-T for 10 minutes each. Protein expression was developed using iBright (FL1000, Thermo fisher scientific, Waltham, MA, USA) by treatment with enhanced chemiluminescence (EzWestLumi plus, ATTO Technology, Tokyo, Japan).

The results are as shown in Figure 3, in the case of pretreatment of 80% ethanol extract (IYC) 10 μg/ml, 20 μg/ml, and 50 μg/ml, VCAM-1, ICAM-1, IL- The expression of 6 decreased in a concentration dependent manner.

From the above examples, it was confirmed that the expression of VCAM-1, ICAM-1, and IL-6 proteins induced by the cytokine TNF-α was reduced by the pretreatment of the Japanese leaf extract.

[Example 4]

Evaluation of mononuclear cell adhesion inhibition

In the present example, the degree of inhibition of the adhesion of monocytes induced by ox-LDL was evaluated by the extract of Japanese chives. After pretreatment of the ethanol extract by concentration, the following adhesion assay was performed to determine the degree of adhesion of HL-60 cells (human leukemia cells) induced by ox-LDL in vascular endothelial cells.

In a 6-well plate, 80% ethanol extract (IYC) was applied to human vascular endothelial cells by concentration for 30 minutes, and then Oxidized Low-Density Lipoprotein (Ox-LDL) was added to a concentration of 50 μg/ml. Incubated for 24 hours. HL-60 cells were treated with BCECF-AM at a concentration of 10 μM and incubated at 37° C. for 1 hour for labeling. HL-60 cells were placed in a plate of HUVEC stimulated by ox-LDL for 24 hours, cultured for 1 hour, and photographed through a fluorescence microscope (Nikon Eclipse Ti, Tokyo, Japan). Fluorescence intensity was measured at excitation and emission wavelengths of 485 nm and 535 nm using a spectrofluorometer (F-2500, Hitachi, Tokyo, Japan).

As a result, as shown in Figs. 4 and 5, when the ox-LDL was treated, the adhesion of HL-60 cells to human vascular endothelial cells was increased, and the degree of adhesion was increased through pretreatment with ethanol extract (IYC) It was confirmed that it decreased. The degree of adhesion showed a tendency to decrease in a concentration-dependent manner at the concentrations of 10, 20, and 50 μg/ml of the ethanol extract of Hanji. In particular, at the concentration of 50 μg/ml, the adhesion level was almost the same as that of the normal group.

From this, it can be seen that the extract of Ichigolite can be useful for hyperlipidemia by inhibiting the adhesion of monocytes induced by oxidized low specific intermediate lipoprotein (ox-LDL) in the blood.

Claims (11)

A pharmaceutical composition for the prevention or treatment of vascular disease, comprising an extract of Japanese peppercorns as an active ingredient.
The pharmaceutical composition of claim 1, wherein the vascular disease is selected from the group consisting of atherosclerosis, coronary artery disease, myocardial infarction, angina, hemangioma, stroke, cerebral infarction, arrhythmia, hypertension, hyperlipidemia, and heart failure. .
The pharmaceutical composition according to claim 1, wherein the vascular disease is hyperlipidemia.
The pharmaceutical composition of claim 1, wherein the vascular disease is atherosclerosis.
The pharmaceutical composition according to claim 1, wherein the monoleaflet extract inhibits the expression of an inflammatory protein selected from the group consisting of VCAM-1, ICAM-1, and IL-6.
The pharmaceutical according to claim 3, wherein the monoleaflet extract exhibits a preventive or therapeutic effect on hyperlipidemia by inhibiting the adhesion of monocytes induced by oxidized low specific gravity protein (ox-LDL) in the blood. Composition.
The pharmaceutical composition according to claim 1, wherein the monoleaflet extract is extracted using C1 to C6 alcohol, water, or a mixture thereof.
The pharmaceutical composition according to claim 1, wherein the monoleaflet extract is extracted using 80% ethanol.
A food composition for preventing or improving vascular disease, comprising an extract of Japanese peppercorns as an active ingredient.
The food composition according to claim 9, wherein the food composition is a health functional food, a dairy product, a fermented product, or a food additive.
Animal feed composition for the prevention or improvement of vascular disease comprising a leaf extract as an active ingredient.

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