KR20090082739A - Composition Comprising Cordycepin for Inhibiting Migration and Proliferation of Vascular Smooth Muscle Cell - Google Patents

Abstract

A composition for inhibiting the movement and proliferation of the vascular smooth muscle cell is provided to use it for treating and preventing the various cardiovascular diseases. A composition for inhibiting the movement and proliferation of the vascular smooth muscle cell comprises cordycepin as an active ingredient. Preferably the cordycepin is separated and purified from Cordyceps militaris. The movement and proliferation of the vascular smooth muscle cell is mediated by the mitogen activation protein kinase or the heat shock protein induced by the platelet-derived growth.

Description

Composition Comprising Cordycepin for Inhibiting Migration and Proliferation of Vascular Smooth Muscle Cell}

The present invention provides a composition for the prevention and treatment of cardiovascular diseases comprising cordycepin , an active ingredient of 3'-deoxyadenosine, isolated from Cordyceps militaris , which is one of several types of Cordyceps sinensis. More particularly, the present invention relates to a pharmaceutical composition for preventing and treating cardiovascular diseases, comprising as a active ingredient a cordycepin having an ability to inhibit vascular smooth muscle cell movement and proliferation signaling and comprising a pharmaceutically acceptable carrier.

In the early 20th century, deaths from cardiovascular disease accounted for less than 10% of all deaths, but recently death rates from cardiovascular disease account for about 50% in developed countries and about 25% in developing countries. Cardiovascular diseases are known to be caused mainly by the proliferation of vascular smooth muscle cells. Specifically, when endothelial cell permeability is increased by injury of vascular endothelial cells, platelet aggregation occurs at the site of injury, and platelet-derived growth factor (PDGF) is released from the aggregated platelets, and the PDGF causes vascular The migration of the smooth muscle cells from the intima to the intima occurs to promote the proliferation and migration of vascular smooth muscle cells. On the other hand, monocyte-derived macrophages present in the blood invade from the injury site of endothelial cells, devour a large amount of fat, and become arterial cells, causing fat accumulation to cause atherosclerosis. This proliferation and hypertrophy of vascular smooth muscle cells is one of the important characteristics of hypertensive patients (Owens, GK, Circ.Res., 56, 525, 1985). In addition, platelet aggregation is an essential step leading to blood coagulation and thrombus formation, and is easily affected by increased physiological activity of platelets or changes in lipid metabolism in plasma.

In order to prevent the progression of atherosclerosis, researches on drugs that inhibit the proliferation of vascular smooth muscle cells have been widely conducted. Several drugs have been used for the treatment of patients (J Am Coll Cardiol., 2002, 39, 183-193). ). However, in the case of drugs currently used, the effect is not perfect, and most of them have a problem of being an organic compound. Therefore, there is a need for the development of new drugs that have the ability to inhibit the migration and proliferation of vascular smooth muscle cells with fewer side effects.

On the other hand, Cordyceps sinensis is a fungus that enters into the body of a live insect at a time when the temperature and humidity of the fungus grow mainly. It is a kind of medicinal mushroom that forms on the epidermis. Around 800 species of Cordyceps fungi are known throughout the world, and among them, Codyceps sinensis is one of the most valuable herbal medicines. It is known to have effects on anti-aging, endocrine, sexual function recovery, respiratory, hepatic circulatory, anticancer and kidney disease (Zhu. JS, J. Altern. Complement. Med., 4. 289-303, 1998). Cordyceps militaris is another species of Cordyceps that is widely used in China and Korea.

Cordycepin is a nucleoside metabolite as one of the main active ingredients of cordyceps. The physiological activities of cordycepin include inhibition of synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), enhancement of cytoskeleton distrinution, cell differentiation, inhibition of bladder cancer, lung cancer and colon cancer. Immune boosting and antibacterial effects are known.

However, there have been no reports on the effects of cordycepin on the migration and proliferation of vascular smooth muscle cells, which are important factors in pathologies such as cardiovascular, cerebrovascular, arteriosclerosis, and hypertension. Therefore, there is a need to develop a material capable of inhibiting the migration and proliferation of superior vascular smooth muscle cells with little side effects and toxicity, such as natural compounds contained in functional foods such as cordyceps.

Therefore, the inventors of the present invention, while searching for various functional foods and traditional medicines for the development of new cardiovascular treatments, cordycepin isolated from cordyceps millitaris strongly inhibits the vascular smooth muscle cell migration and proliferative signal transduction and hypertension and arteries. The present invention has been completed by confirming that the endothelial cell damage, which is very important for the development of cardiovascular diseases such as hardening, and thereby effectively controlling the proliferation and migration of vascular smooth muscle cells.

After all, the main object of the present invention is to provide a composition for inhibiting the movement and proliferation of vascular smooth muscle cells containing cordycepin as an active ingredient.

In addition, another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of cardiovascular diseases comprising the composition and a pharmaceutically acceptable carrier.

In order to achieve the above object, the present invention provides a composition for inhibiting the movement and proliferation of vascular smooth muscle cells containing cordycepin as an active ingredient.

In the present invention, the cordycepin may be characterized in that it is separated and purified from cordyceps millitaris, the migration and proliferation of the vascular smooth muscle cells are platelet-derived growth factor (PDGF) By mitogen activated protein kinase (p38 MAPK) or heat shock protein (HSP 27).

The present invention also provides a pharmaceutical composition for the prevention or treatment of cardiovascular diseases comprising the composition containing the cordycepin as an active ingredient and a pharmaceutically acceptable carrier.

In the present invention, the cardiovascular disease is atherosclerosis, hypertension, angina pectoris, cerebral infarction. Myocardial infarction and heart failure may be selected from the group consisting of.

Cordycepin of the present invention inhibits vascular smooth muscle cell migration and proliferation signaling processes mediated by platelet-derived growth factor (PDGF) -induced mitogen-activated protein kinase (p38 MAPK) and heat shock protein (HSP 27). Since it effectively regulates damage to vascular endothelial cells and thereby the movement and proliferation of vascular smooth muscle cells, which are important for the development of angina, arteriosclerosis, hypertension, and cerebral infarction, they can be usefully used for the prevention and treatment of various cardiovascular diseases.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for inhibiting migration and proliferation of vascular smooth muscle cells containing cordycepin as an active ingredient.

Cordycepin according to the present invention can inhibit the migration and proliferation signaling process of vascular smooth muscle cells mediated by platelet-derived growth factor-induced mitogen-activated protein kinase and heat shock protein.

That is, concentration-dependent inhibition of vascular smooth muscle cell migration and proliferation induced by platelet-derived growth factor, especially vascular smooth muscle cell migration was very strongly inhibited at 10 ㎍ / ㎖ of cordycepin, and proliferation of vascular smooth muscle cell It was found that inhibition was excellent at 0.1 g / ml of cordycepin.

In addition, the present invention examines the effect of cordycepin on mitogen-activated protein kinase and heat shock protein signaling during the migration and proliferation of vascular smooth muscle cells. It was found to inhibit the migration and proliferation of mediated vascular smooth muscle cells.

In the present invention, the cordycepin may be used commercially available or can be used to separate and purified from cordyceps millitaris, preferably can be used to separate and purify from cordyceps millitaris.

These cordycepins can be widely used for the prevention and treatment of cardiovascular diseases because they excellently inhibit the signaling processes associated with the migration and proliferation of vascular smooth muscle cells, which are very important for the development of cardiovascular diseases such as hypertension, arteriosclerosis, and angina pectoris. .

The present invention provides a pharmaceutical composition for preventing or treating cardiovascular disease, comprising a composition for inhibiting migration and proliferation of vascular smooth muscle cells containing cordycepin as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be used independently or as pharmaceutical additives with a pharmaceutically acceptable carrier or excipient according to the application or in any other form suitable for administration to humans. Carriers that may be contained in the pharmaceutical compositions of the present invention may include extenders, high fiber additives, encapsulating agents, lipids, and the like, examples of which are well known in the art.

The pharmaceutical composition of the present invention may be administered in varying dosages depending on the extent of disease, age, sex, physical condition, duration of administration, method of administration, weight of the patient, diet, rate of discharge, and the like.

However, parenteral or oral administration of the pharmaceutical composition of the present invention in a range of 0.01-100 μg / kg body weight per day for the desired effect is preferred. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation and Identification of Cordycepin

Cordyceps Militaris was used by drying what was purchased at Gyeongdong Market (Seoul, Korea). The dried Codycept millitaris was ground in a grinder, and then powdered. 10 ml / g of 80% ethanol was added to 1 gram of powder, and the mixture was refluxed with reflux for 3 hours at 80 ° C. for 3 hours. To prepare an ethanol crude extract.

The crude ethanol extract concentrate was dissolved in distilled water, butanol was mixed at a ratio of 1: 1 (v / v), and the mixture was repeated three times. The butanol extract was concentrated under reduced pressure, concentrated, and dried to give a dark brown powder. To the powder, cordycepin fractions obtained by performing silica gel column chromatography (solvent type: CHCl ₃ -EtOAc-MeOH = 12: 1: 3) were decompressed, dried again and then dissolved in methanol to crystallize into pure white or transparent needles. Cordycepin crystals (needle type, single crystal) were obtained.

Cordycepin obtained as described above was found to have a purity of 100% as a result of a purity test (three different 100% HPLC methods) according to the standard analysis method prescribed by the International Harmonized Committee. Thus it can be proved that the activity is not due to any impurity that may be contaminated.

Experimental Example 1 Investigation of the Effect of Cordycepin on the Migration of Vascular Smooth Muscle Cells

In order to investigate the effects of cordycepin of the present invention on platelet-derived growth factor (PDGF) -induced vascular smooth muscle cell migration, the following experiments were performed.

First, PDGF induced the migration of vascular smooth muscle cells, and the results were measured using a Boyden chamber assay. The migration of these vascular smooth muscle cells increased concentration-dependently with PDGF, especially when the PDGF concentration was 10 ng / ㎖ showed a strong migration effect (see Figure 1A). The vascular smooth muscle cell migration induced by 10 ng / ml of PDGF was increased by 196.35 ± 12.7% compared to the control group not treated with PDGF, and PDGF (10 ng / ml) induced vascular smooth muscle cell migration by cordycepin. Was inhibited in a concentration-dependent manner, particularly at 10 μg / ml (see FIG. 1B).

Experimental Example 2: Investigation of the effect of cordycepin on vascular smooth muscle cell proliferation

In order to investigate the effect of cordycepin of the present invention on the proliferation of vascular smooth muscle cells induced by platelet-derived growth factor (PDGF), the concentration of cordycepin described in Experimental Example 1 was investigated as follows.

First, PDGF induced the proliferation of vascular smooth muscle cells, and the results were measured using the Bromodeoxyuridine assay (BrdU assay). The proliferation of these vascular smooth muscle cells was increased concentration-dependently by PDGF, especially when the PDGF concentration is 10 ng / ㎖ showed a strong proliferation effect (see Figure 2A). Proliferation of 10 ng / ml PDGF-induced vascular smooth muscle cells increased to 190.39 ± 10.7% compared to the control group not treated with PDGF, and PDGF (10 ng / ml) induced vascular smooth muscle cell proliferation It was confirmed that the concentration was inhibited by cepin and strongly inhibited at 0.1 μg / ml of cordycepin (see FIG. 2B).

Experimental Example 3: Toxicity of Cordycepin to Vascular Smooth Muscle Cells

In order to confirm the toxicity of cordycepin to vascular smooth muscle cells, the present invention was investigated as follows using cordycepin at the concentrations used in Experimental Examples 1 and 2.

First, cordycepin was treated to vascular smooth muscle cells and measured by XTT assay. The viability of these vascular smooth muscle cells showed more than 75.07 ± 6.8% at the concentration of cordycepin at 0.01-1 μg / ml, whereas it showed 8.16 ± 0.52% at the concentration of 10 μg / ml ( 3).

Experimental Example 4: Investigation of the effects of cordycepin on p38 MAPK and HSP 27 signaling

In order to investigate the effects of cordycepin of the present invention on p38 MAPK and HSP 27 signaling in the process of vascular smooth muscle cell migration and proliferation, it was performed as follows.

First, as a result of investigating the effects of cordycepin on PDGF-induced p38 MAPK and HSP 27 activation, PDGF (10 ng / ml) increased the phosphorylation of p38 MAPK by 237.18 ± 25.18% compared to the control group (Fig. 4). A and B), cordycepin 1, 10 μg / ml inhibited phosphorylation of p38 MAPK up to 98.26 ± 23.62% and 73.35 ± 16.94%. In addition, PDGF (10 ng / ml) increased the phosphorylation of HSP 27 to 267.23 ± 29.6% and reduced phosphorylation by 204.71 ± 38.03% and 156.57 ± 34.03% at 1 and 10 μg / ml, respectively, compared to the control. It could be confirmed (A and C of Fig. 4). In contrast, PDGF (10 ng / ml) increased the phosphorylation of P-Erk 1/2, but did not affect the phosphorylation of Erk 1/2 (A and D in FIG. 4).

Experimental Example 5: Investigation of the effect of cordycepin on the migration and proliferation of vascular smooth muscle cells

The present invention was performed as follows to investigate the effect of cordycepin on the movement and proliferation of smooth muscle cells forming blood vessels.

First, the aorta was extracted from the white paper, and only the smooth muscle layer from which vascular endothelial cells and outer membrane fibrous cells were removed was isolated. Then, the smooth muscle cell migration and proliferation from the separated aortic smooth muscle layer were confirmed using PDGF.

It was confirmed that PDGF-induced migration and proliferation of aortic smooth muscle cells (217.03 ± 6.79%) was suppressed to 161 ± 21.26% to 37.96 ± 6.2% in cordycepin 1 to 10 ㎍ / ml of the present invention (Fig. 5). A and B).

1 is a graph showing the effect of cordycepin and platelet-derived growth factors of the present invention on the movement of vascular smooth muscle cells.

2 is a graph showing the effect of cordycepin and platelet-derived growth factors of the present invention on the proliferation of vascular smooth muscle cells.

3 is a graph showing the effect of cordycepin of the present invention on the toxicity of vascular smooth muscle cells.

4 is a graph showing the effect of cordycepin of the present invention on mitogen-activated protein kinase and heat shock protein activity induced by platelet-derived growth factors.

5 is a graph showing the effect of cordycepin of the present invention on the response of vascular smooth muscle cells induced by platelet-derived growth factors.

Claims (6)

Composition for inhibiting the movement and proliferation of vascular smooth muscle cells containing cordycepin as an active ingredient. The composition of claim 1, wherein the cordycepin is isolated and purified from Cordyceps militaris cordyceps. 2. The composition of claim 1, wherein the migration and proliferation of vascular smooth muscle cells is mediated by mitogen-activated protein kinases or heat shock proteins induced by platelet-derived growth factors. A pharmaceutical composition for preventing or treating cardiovascular disease, comprising the composition of claim 1 and a pharmaceutically acceptable carrier. The method of claim 4, wherein the cardiovascular disease is atherosclerosis, hypertension, angina pectoris, cerebral infarction. Composition for the prevention and treatment of cardiovascular diseases, characterized in that selected from the group consisting of myocardial infarction and heart failure. According to claim 4, wherein the pharmaceutical composition is a composition for the prevention and treatment of cardiovascular diseases, characterized in that it further comprises a pharmaceutically acceptable excipient or therapeutic aid.

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