KR20230091467A - Composition for preventing or treating of vascular calcification containing extracts of platycarya strobilacea - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of vascular calcification comprising an extract of cinnamon tree as an active ingredient. can be used

Description

Composition for preventing or treating vascular calcification containing an extract of cinnamon tree

The present invention relates to a pharmaceutical composition for preventing or treating vascular calcification and a pharmaceutical composition for preventing or treating a disease related to vascular calcification, including a cinnamon tree extract.

Blood vessels are organs that continuously circulate body fluids, supplying oxygen and nutrients to tissues, and receiving and processing carbon dioxide or waste products from tissues. Vascular calcification is the deposition of minerals such as calcium and phosphoric acid in blood vessels, which increases the stiffness of blood vessels and causes blood vessel rupture. Such vascular calcification is commonly found in patients with arteriosclerosis, diabetes, and chronic renal failure, and loss of calcium accumulation inhibitors, induction of bone formation, nucleation complex in the blood, and cell death are considered to be the main causes of vascular calcification, but the exact mechanism has not yet been revealed.

In particular, cardiovascular calcification contributes to the aggravation of hypertension, heart failure, acute coronary syndrome and valve disease, and causes various complications (sudden cardiac death, myocardial infarction, angina pectoris, ischemic heart failure, etc.). Also, in postmenopausal women, vascular calcification occurs in addition to osteoporosis. However, since the precise mechanism of vascular calcification has not yet been identified, preventive and therapeutic agents have not yet been developed. Therefore, it is necessary to develop a technique for preventing, improving or treating vascular calcification.

1. Domestic Patent No. 10-2331735

In the above circumstances, the inventors of the present invention completed the present invention by confirming that, as a result of research to find a natural product-derived vascular calcification inhibiting substance, the extract of oyster extract inhibits the accumulation of calcium in vascular cells without cytotoxicity.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating vascular calcification, and a health functional food composition for preventing or improving vascular calcification, comprising a cinnamon tree extract as an active ingredient.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating vascular calcification comprising a cinnamon tree extract as an active ingredient.

In the present invention, "prevention" means any action to inhibit vascular calcification or delay its progression by administration of the pharmaceutical composition according to the present invention.

In the present invention, "treatment" refers to all activities that improve or beneficially change the symptoms of vascular calcification by administration of the pharmaceutical composition according to the present invention.

Platycarya strobilacea ) is a deciduous broad-leaved tree belonging to the genus Gyrusaceae, and is distributed in the middle of the mountain in the middle and south of Korea. In oriental medicine, the fruits and roots are used as anti-inflammatory and anti-diarrheal agents, but the efficacy related to the inhibition of vascular calcification is unknown.

As used herein, the term "extract" refers to a preparation obtained by squeezing an extraction target into an appropriate leachate and concentrating or filtering the leachate, but is not limited thereto, an extract obtained by extraction treatment, a dilution or concentrate of the extract, It may be a dried product obtained by drying the extract, a crude purified product or a purified product thereof.

The cinnamon tree extract can be prepared using a general extraction method, separation and purification method known in the art. The extraction method is not limited thereto, but hot water extraction, hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction may be used.

In the present invention, the extraction solvent may be selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, dichloromethane, acetone, and mixed solvents thereof, preferably methanol.

In one embodiment of the present invention, an extract prepared by immersing the dried stem part of the cinnamon tree in methanol, repeating sonication, and drying under reduced pressure was used (Example 1).

On the other hand, these cinnamon tree extracts may be included in 0.001 to 50% by weight based on the total weight of the composition, preferably 0.05 to 20% by weight.

Vascular calcification means that minerals such as calcium and phosphoric acid are deposited in the blood vessels to increase the stiffness of the blood vessels, which may result in rupture of the blood vessels. In the present invention, vascular calcification includes calcification of coronary artery, aorta and other blood vessels, and includes intimal calcification of vessel wall, septal calcification, heart valve calcification, and resistant calcium formation (calcific uremic arteriolopathy).

Intimal calcification is associated with dyslipidemia, inflammation and intimal thickening, leading to atherosclerosis, which results in occlusive vascular disease by forming unstable and ruptured plaques on the inner vessel walls. In contrast, medial calcification (Mφnckeberg's arteriosclerosis), which is common mainly in diabetes or chronic kidney disease, is known to occur when bone formation-stimulating factors are expressed in blood vessels due to diabetes, abnormal calcium metabolism, or renal disease. Since it can occur even without vascular lumen stenosis, it mainly causes vascular stiffness and increases the occurrence of cardiovascular disease complications.

In the present invention, the vascular calcification may be caused by osteoporosis, hyperlipidemia, aging, kidney disease, hypertension, cardiovascular disease, or thrombosis.

According to one embodiment of the present invention, the cumin tree extract inhibits the deposition of calcium in vascular cells, and also inhibits the activation of osteoclasts. Therefore, the composition further exhibits not only vascular calcification inhibitory activity but also osteoclast differentiation inhibitory activity.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is one commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, skin application, intravenous, subcutaneous, intraperitoneal injection or topical application) depending on the desired method, but oral administration is preferred.

When the active ingredient of the present invention is formulated into tablets, capsules, chewing tablets, powders, solutions, suspensions, etc. for the purpose of oral administration, binders such as gum arabic, corn starch, microcrystalline cellulose or gelatin, phosphoric acid Excipients such as dicalcium or lactose, disintegrants such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sweeteners such as sucrose or saccharin, and flavoring agents such as peppermint, methyl salicylate or fruit flavors may be included. there is.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, 'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. For example, the pharmaceutical composition of the present invention can be administered at a dose of 0.1 mg/kg to 200 mg/kg, preferably 1 mg/kg to 50 mg/kg once a day, or divided into three times a day. there is. The dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Another aspect of the present invention provides a health functional food composition for preventing or improving vascular calcification comprising a cinnamon tree extract as an active ingredient.

Since the health functional food composition for preventing or improving vascular calcification of the present invention uses the gulphicus extract described in the above-described pharmaceutical composition, the common content between the two is to avoid excessive complexity of the present specification, the description omit

The functional food composition of the present invention may be used alone or in combination with other foods or food ingredients, and may also be appropriately used according to conventional methods. The mixing amount of the cinnamon tree extract may be suitably determined according to its purpose of use (for prevention or improvement). In general, 15% by weight or less, preferably 0.0001 to 10% by weight, based on the total weight of the composition of the present invention may be added during the preparation of food or beverage, but for health and hygiene purposes or for health control In the case of long-term intake to be, the amount may be less than the above range.

In addition to containing the above active ingredients as essential components, the functional food composition of the present invention contains carbohydrate foods (cereals, legumes, pulses, sugar, and taffy, etc.), protein foods (meat, eggs, milk, fish and shellfish, soybeans, and Peanuts, etc.), oil-and-fat foods (vegetable oil, animal fat, and margarine, etc.) may be provided. In addition, vitamin foods (green and yellow vegetables, fruits, liver, fish and shellfish, etc.) and mineral foods (bone-eating small fish, seaweed, salt, milk, etc.) may be additionally contained as needed.

In addition to the above, the functional food composition of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. These components may be used independently or in combination. The ratio of these nutrients and the like can also be appropriately selected by those skilled in the art.

Since the cumin tree extract according to the present invention has an effect of inhibiting the accumulation of calcium in vascular cells without cytotoxicity, it can be usefully used for preventing or treating vascular calcification.

Figure 1 shows the result of intracellular calcium staining (A) and the result of quantifying the degree of staining (B) after treatment with oyster extract while differentiating human arterial smooth muscle cells into calcified cells.
Figure 2 is a result of observing the cells under a microscope (A) and quantifying the actual calcium concentration in the cells (B) after treating the cells with the extract of the oyster tree while differentiating human arterial smooth muscle cells into calcified cells.
Figure 3 is the result of confirming the cell proliferation level (A) and cytotoxicity level (B) after treatment with the extract of oyster tree while differentiating human arterial smooth muscle cells into calcified cells.
Figure 4 is a result of TRAP staining after treatment with oyster extract while differentiating macrophages into osteoclasts (A) and the ratio of TRAP+ osteoclasts having 3 or more or 10 or more nuclei in one cell (B) is the result of checking
Figure 5 is the result of measuring the activity of osteoclasts after treating with the extract of oyster tree while differentiating macrophages into osteoclasts.
Figure 6 is the result of confirming the level of cell proliferation after treating the extract of oyster tree extract while differentiating macrophages into osteoclasts.
Figure 7 is the result of confirming the level of cytotoxicity after treating the extract of oyster tree while differentiating macrophages into osteoclasts.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of cinnamon tree extract

Olympus cinnamon stem extract (KPM042-014) was obtained from the Korea Plant Extract Bank (Korea Plant Extract Bank; Daejeon, Korea) under the Korea Research Institute of Bioscience and Biotechnology (KRIBB; Daejeon, Korea).

The extract was prepared as follows. After drying the stems of oyster trees (sample number: KRIB 0083802) collected in Seogwipo-si, Jeju-do in 2017 in the shade, 96 g of the dried stems were put into 1 liter of methyl alcohol (99.9%, HPLC grade), and then placed in an ultrasonicator at room temperature. (SDN-900H, SD-ULTRASONIC CO., LTD) the following steps were repeated 30 times: 40 KHz, 1500 W, 15 minutes per cycle. Ultrasonification 120 minutes. Thereafter, filtered (Qualitative Filter No.100, HYUNDAI MICRO CO., LTD) and dried under reduced pressure to obtain a cumin extract (6.6 g).

Example 2: Inhibiting vascular calcification effect of cinnamon tree extract

2-1. Alizarin red staining and quantification

The effect of the extract of sycamore tree on the calcification in the atherosclerotic plaque was confirmed as follows. An increase in calcification is associated with an increase in cardiovascular events.

Human aortic smooth muscle cells, which are vascular cells capable of differentiating into calcified cells by pathological stimuli in the artery, were seeded on a plate and treated with a calcified cell differentiation medium (including angiotensin II) for 14 days. Culphia extract was treated at a concentration of 5, 10 or 20 μg/ml every 3 days.

After 14 days, cells were stained with alizarin red capable of staining calcium to check the degree of calcification, and alizarin red stained with 10% acetic acid was extracted to measure the concentration. Data are expressed as mean ± standard error. As a result, it was confirmed that calcification was inhibited depending on the treatment concentration in the oyster extract treatment group compared to the control group not treated with the oyster extract (Fig. 1; p***<0.001, p**<0.01) .

2-2. Calcium assay

As a result of quantifying the actual intracellular calcium concentration, it was found that the intracellular calcium concentration decreased depending on the treatment concentration in the oyster extract treatment group compared to the control group (Fig. 2; p***<0.001, p** <0.01).

2-3. WST assay (cell viability assay) and LDH assay (cell cytotoxicity assay)

Human aortic smooth muscle cells, which are vascular cells capable of differentiating into calcified cells by pathological stimuli within the artery, are dispensed into plates, and calcified by treatment with calcified cell differentiation medium (including angiotensin II) for 14 days. Differentiation into cells was induced. Culphia extract was treated at a concentration of 5, 10 or 20 μg/ml every 3 days.

After 14 days, tetrazolium salt of EZ-Cytox Kit (DoGenbio, Korea) was added to the cell culture medium to evaluate the cell proliferation ability, and absorbance was measured at 450 nm. As a result of the measurement, the number of cells slightly decreased when the extract of 20 ㎍ / ㎖ was treated with calcification medium, but since there is a phenomenon in which the increase in the number of cells slows down when human arterial smooth muscle cells are differentiated by the calcification medium, this decrease is not a significant difference. (Fig. 3A; p*<0.005).

In addition, as a result of measuring lactate dehydrogenase (LDH) released from cells after 14 days with Lactate dehydrogenase (LDH) Cytotoxicity Detection Kit (Takara Bio Inc, Japan), there was little difference between the control group and the oyster extract treatment group. It was confirmed (Fig. 3B).

Example 3: Inhibitory effect of Osteoclast Activation of Osteoclast Extract

3-1. TRAP staining

In a 48-well plate, RAW264.7 cells, a mouse macrophage cell line, were dispensed 3x10 ³ per well, and RANKL (receptor activator of NK-κB ligand), an osteoclast differentiation inducer, was treated at a concentration of 40 ng/ml to induce differentiation did Afterwards, the medium was cultured for a total of 4 days while replacing the medium every 2 days, and when the medium was replaced, the oyster extract was treated together.

After 4 days, the differentiated cells were fixed with a fixative for 5 minutes at room temperature and washed several times with distilled water. A substrate solution of sodium tartrate and acid phosphatase was mixed, and cells were treated with the prepared mixture to stain tartrate-resistant acid phosphate (TRAP), an osteoclast differentiation marker. Staining was performed at 37° C. for 15 to 45 minutes after covering the plate with a lid to prevent drying. Thereafter, the dye was discarded and washed three times with distilled water to stop the reaction. After observation under a microscope, pictures were taken and the number of stained cells was counted using the Image J program.

As a result of the experiment, it was found that differentiation of osteoclasts was suppressed in the oyster extract treatment group compared to the control group treated with only RANKL. Specifically, the activity of osteoclasts was found to dissolve calcium-phosphate, which is the matrix of bone. In FIG. 4A, white holes indicate that osteoclasts were activated and dissolved the matrix. It can be seen that the number and average area of the white pores increased by the RANKL treatment decreased and became smaller by the Kyrgyzalis extract treatment (Fig. 4A).

In addition, since osteoclasts are characterized by having a plurality of nuclei, the ratio of TRAP ⁺ osteoclasts having 3 or more (N≥3) or 10 or more (N≥10) nuclei in one cell was calculated. As a result, compared to the control group treated only with RANKL, the ratio of TRAP ⁺ osteoclasts having 3 or more (N≥3) or 10 or more (N≥10) nuclei was significantly reduced in the oyster extract treatment group. confirmed (FIG. 4B).

3-2. Pit assay

Bone resorption assay kit (CSR-BRA-48KIT, COSMOBIO) was used to confirm the activity of RAW264.7 cells differentiated into osteoclasts by RANKL treatment. Specifically, RAW264.7 cells were seeded onto a culture dish coated with a bone-mimicking mattress containing a calcium-phosphate layer, and differentiation was induced by treatment with RANKL, an osteoclast differentiation inducer, at a concentration of 40 ng/ml. Afterwards, the medium was cultured for a total of 4 days while replacing the medium every 2 days, and when the medium was replaced, the oyster extract was treated together. After 6 days, osteoclasts attached to the culture dish were removed by treatment with 5% sodium hypochlorite for 5 minutes. The petri dish was further washed with water and then dried. After taking a picture of each well, the pit area formed by osteoclasts decomposing the bone-like mattress was measured with Image J. The degradation area is proportional to the activity of osteoclasts.

As a result of the measurement, compared to the control group treated only with RANKL, it was confirmed that the decomposition area was reduced when the oyster extract treatment group was treated at concentrations of 5 and 10 μg / ml (FIG. 5).

3-3. WST assay (cell viability assay)

When macrophages are treated with RANKL, they are differentiated and activated into osteoclasts, so the number of cells does not increase, and three or more cells coalesce with each other. Therefore, when the WST assay based on cellular mitochondrial activity is performed, the total measured value decreases in the RANKL-treated group compared to the control group regardless of cell death. can

However, when RAW264.7 cells were treated with RANKL and oyster extract, it was confirmed that the effect of RANKL was offset by oyster extract (FIG. 6).

3-4. LDH assay (cell cytotoxicity assay)

In addition, LDH assay was performed to measure lactate dehydrogenase (LDH) released from cells after RAW264.7 cells were treated with RANKL and oyster extract. As a result, it was confirmed that both RANKL and cinnamon extract had no cytotoxicity (FIG. 7).

Claims (8)

A pharmaceutical composition for the prevention or treatment of vascular calcification comprising a cinnamon tree extract as an active ingredient. According to claim 1, wherein the cinnamon tree extract is extracted with a solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, dichloromethane, acetone, and mixed solvents thereof, for preventing or treating vascular calcification. enemy composition. According to claim 2, wherein the cumin tree extract is a methanol extract, a pharmaceutical composition for the prevention or treatment of vascular calcification. According to claim 1, wherein the cinnamon tree extract is a cinnamon tree stem extract, a pharmaceutical composition for the prevention or treatment of vascular calcification. The pharmaceutical composition for preventing or treating vascular calcification according to claim 1, wherein the composition further has an osteoclast differentiation inhibitory activity. According to claim 1, wherein the vascular calcification is osteoporosis, hyperlipidemia, aging, renal disease, hypertension, cardiovascular disease or thrombosis induced by, preventive or therapeutic pharmaceutical composition for vascular calcification. A health functional food composition for preventing or improving vascular calcification comprising a cinnamon tree extract as an active ingredient. According to claim 7, wherein the cinnamon tree extract is a methanol extract, a functional health food composition for preventing or improving vascular calcification.

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