KR102628997B1 - Composition Comprising the Extract of Senna Alata for Preventing or Inhibiting Vascular Aging - Google Patents

Abstract

The present invention provides a food composition for preventing or suppressing vascular aging, or preventing or improving cardiovascular disease, containing Senna alata extract as an active ingredient; Health functional food containing the above food composition; and a pharmaceutical composition for preventing or treating cardiovascular disease containing Senna allata extract as an active ingredient.
The Senna allata extract according to the present invention inhibits senescence associated beta galactosidase (SA-β-gal) activity and reduces the expression of aging-related proteins (p16, p21, p53, and PAI-1), thereby excellently suppressing vascular cell aging. Because of its effectiveness, a composition containing Senna alata extract can not only be used to prevent or inhibit vascular aging, but can also be usefully used in the manufacture of foods and medicines for preventing, improving, or treating cardiovascular diseases.

Description

Composition for preventing or inhibiting vascular aging comprising Senna Alata extract {Composition Comprising the Extract of Senna Alata for Preventing or Inhibiting Vascular Aging}

The present invention provides a food composition for preventing or suppressing vascular aging, or preventing or improving cardiovascular disease, containing Senna alata extract as an active ingredient; Health functional food containing the above food composition; and a pharmaceutical composition for preventing or treating cardiovascular disease containing Senna allata extract as an active ingredient.

Vascular endothelial cells are a layer of cells that line the vascular lumen and constitute the intima. They play a major regulatory role in maintaining vascular homeostasis, including vascular expansion and contraction, proliferation and migration of vascular smooth muscle, and thrombus formation and dissolution. Dysfunction of vascular endothelial cells disrupts this balance, resulting in damage to the artery wall and can be used as an early marker for atherosclerosis. Endothelial dysfunction may occur during the aging process.

In aged vascular endothelial cells, the production of nitric oxide, the expression of eNOS (endothelial nitric oxide synthase), and the production of prostacyclin decrease, resulting in significantly lower vascular contractility, and PAI-1 (type I plasminogen activator) The expression increases, promoting thrombus formation, and the expression of cell adhesion proteins and chemokines such as Inter-Cellular Adhesion Molecule 1 (ICAM-1), interleukin-1, and interleukin-8 increases, resulting in increased inflammatory response and narrowing of blood vessels. You lose. In fact, vascular endothelial cells in atherosclerotic lesion tissue are known to have increased SA-β-Gal (senescence associated beta-galactosidase) activity, a well-known marker of cellular aging, compared to cells in normal tissue.

Since senescent cells do not undergo smooth cell division, when senescent cells accumulate, not only damaged tissues cannot be quickly repaired, but they also secrete enzymes or inflammatory cytokines that decompose surrounding tissues, thereby accelerating tissue damage, which is related to aging. Contributes to the pathogenesis of the disease. In other words, it is believed that inhibiting and delaying the aging of vascular endothelial cells can prevent and treat cardiovascular diseases caused by it.

Since cellular aging is believed to be an essential causal factor for aging, there have been efforts to develop methods to delay cellular aging. Among them, some studies have been reported to explore substances that can control cellular aging and use them to prevent and treat diseases.

Meanwhile, Senna alata is a plant distributed in tropical and wet areas and is known to exhibit various pharmacological activities in its flowers, roots, leaves, and seeds. Specifically, the antibacterial, anti-fungal, and antiviral activities of Senna alata have been reported, and it has been reported to be used in the treatment of wounds, skin infections, allergies, diarrhea, etc. However, there are no examples of using Senna allata extract to suppress cellular aging or apply it to the prevention or treatment of cardiovascular disease.

Under this background, the present inventors found that Senna alata extract has the effect of inhibiting senescence associated beta galactosidase (SA-β-gal) activity and reducing the expression of aging-related proteins (p16, p21, p53, and PAI-1). By identifying, the present invention was completed by confirming that Senna alata extract can be used as a material for food compositions and pharmaceutical compositions to prevent or inhibit vascular aging, or to prevent, improve, or treat diseases related to vascular aging. .

Republic of Korea Patent No. 10-1522596 Republic of Korea Patent No. 10-1816601

The object of the present invention is to prevent or inhibit vascular aging comprising Senna alata extract as an active ingredient; Alternatively, a food composition for preventing or improving cardiovascular disease is provided.

Another object of the present invention is to provide a health functional food containing the above food composition.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular disease containing Senna allata extract as an active ingredient.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

In one aspect for achieving the above object, the present invention provides a food composition for preventing or suppressing vascular aging, or preventing or improving cardiovascular disease, containing Senna alata extract as an active ingredient; And it provides a health functional food containing the above food composition.

The term "extract" in the present invention refers to an extract, such as an extract obtained by extraction treatment of Senna alata, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or a mixture thereof. It includes extracts of all formulations that can be formed on their own and using extracts.

The extract of the present invention may be extracted from the stem, root, leaf, flower, seed, whole plant of Senna alata, or a combination thereof, and specifically, may be extracted from the whole plant of Senna alata, that is, the whole plant.

The method for extracting the extract of the present invention is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, cold immersion extraction, solvent extraction, steam distillation, elution, compression, ultrasonic extraction, filtration, and reflux extraction, which are performed alone or using a combination of two or more methods. It can be performed by doing this.

The extract of the present invention is extracted from Senna alata using an appropriate solvent, and may include, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. The type of extraction solvent used to prepare the extract is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the extraction solvent include water; lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, propyl alcohol, and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; Hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof can be used. Specifically, the extraction solvent may be methanol. Additionally, a solvent extract can be prepared by extracting the extract one or more times using the solvent.

Specifically, in one embodiment of the present invention, the Senna alata extract is obtained by adding 100 to 300 ml of methanol to 20 to 50 g of dried and pulverized Senna alata, and dissolving 10 to 30 methanol under conditions of 40 to 60°C and 1200 to 1800 psi. It may have been extracted using an automatic extractor for several minutes. In addition, in another embodiment of the present invention, the Senna alata extract is prepared by adding 1 to 2 L of methanol to 100 to 300 g of dried and ground Senna alata and using a sonicator for 2 to 4 days at 40 to 60 ° C. It may have been extracted.

The extract of the present invention is filtered to remove floating solid particles, for example, by filtering the particles using nylon, etc., or filtered using filter paper, cryofiltration, etc., and then used as is, or concentrated or dried (e.g., freeze-dried, It can be used by hot air drying, spray drying, etc.). Additionally, the extract may further undergo a conventional fractionation process or may be purified using a conventional purification method.

Specifically, in the present invention, Senna alata may be extracted with a methanol solvent, the extract liquid may be filtered, and the methanol may be evaporated to concentrate the extract for use, but is not limited thereto.

The term "active ingredient" in the present invention refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

The term "cardiovascular disease" in the present invention refers to a general term for vascular diseases, heart diseases, or cerebrovascular diseases of the circulatory system, and specifically refers to arteriosclerosis, hyperlipidemia, hypertension, angina, myocardial infarction, stroke, cardiac arrhythmia, It may be selected from the group consisting of heart failure, endocarditis, ischemic cerebrovascular disease, and ischemic heart disease, but is not limited thereto.

The term "vascular disease of the circulatory system" in the present invention refers to a general term for cases where diseases occur in various organs of the large and small circulatory systems, including the heart, which are involved in blood circulation.

The term "heart disease" in the present invention refers to diseases related to the heart, which is responsible for supplying blood to the human body, and includes ischemic heart disease, cardiac arrhythmia, heart failure, etc. Ischemic heart disease, which typically includes angina pectoris and myocardial infarction, refers to an acute or chronic heart disorder that occurs because blood supply to the myocardium is reduced or stopped as coronary artery disease progresses. Other heart diseases include endocarditis, cardiac arrhythmia, or heart failure. In particular, heart failure refers to a condition in which the heart cannot sufficiently rescue blood returning to the heart through the venous system.

The term "cerebrovascular disease" in the present invention refers to a disease that occurs suddenly due to an abnormality in the cerebrovascular system, causing brain dysfunction and leading to collapse. Depending on the form of onset, the term "cerebrovascular disease" refers to hemorrhagic disease in which blood vessels within the two vessels break and bleed, depending on the form of onset. It can be classified as ischemic cerebrovascular disease in which blood flow is impaired or blocked, but is not limited to this.

In the present invention, the cardiovascular disease may be caused by vascular aging, and specifically, as the patient ages, the endothelial cells that make up the blood vessels age and functional abnormalities accumulate in the process.

In the present invention, the composition can inhibit aging of vascular cells by reducing the activity of senescence associated beta galactosidase (SA-β-gal), an aging marker, in aged vascular cells.

In the present invention, the composition inhibits aging of vascular cells and normalizes the function of vascular cells by reducing the expression of aging markers p16, p21, p53, and PAI-1 (plasminogen activator inhibitor-1) in aged vascular cells. You can. The p53, p16, and p21 are proteins that inhibit cell growth or cell division, and the PAI-1 protein increases inflammation, promotes aging, and promotes the formation of blood clots.

The term “prevention” in the present invention refers to any action that inhibits or delays vascular aging or cardiovascular disease by ingesting the food composition of the present invention.

The term "improvement" in the present invention refers to any action that improves or benefits vascular aging or cardiovascular disease conditions by ingesting the food composition of the present invention.

SA-β-gal, p16, p21, p53, and PAI-1 are known as aging markers, and the correlation between vascular aging and cardiovascular disease is widely known in the art ( J Atheroscler Thromb, 2020; 27: 47-59 and Experimental Gerontology , 2005; 40: 634-642). Therefore, the composition containing the Senna alata extract according to the present invention as an active ingredient inhibits SA-β-gal activity and reduces the expression of aging-related proteins (p16, p21, p53, and PAI-1), thereby producing excellent vascular cells. Since it has an anti-aging effect, it is clear that the composition containing the Senna allata extract has an effect of preventing or suppressing vascular aging and preventing, improving, or treating cardiovascular diseases related thereto.

The food composition of the present invention prevents or inhibits vascular aging; Alternatively, the Senna alata extract may be included in various amounts as long as it has a preventive or ameliorating effect on cardiovascular disease.

In addition to the Senna alata extract, the food composition of the present invention may further include any compound or natural extract whose safety has already been verified in the art and known to have the corresponding activity in order to improve the convenience of taking or ingesting. there is.

These compounds or extracts include compounds, extracts, and medicinal products listed in compendiums such as the Pharmacopoeia of each country ('Korean Pharmacopoeia' in Korea) and the Code of Health Functional Foods of each country ('Standards and Specifications for Health Functional Foods' notified by the Ministry of Food and Drug Safety in Korea). The laws of each country governing the manufacture and sale of compounds, extracts, and health functional foods that have received product approval in accordance with the laws of each country (the “Pharmaceutical Affairs Act” in Korea) (the “Act on Health Functional Foods” in Korea). Accordingly, compounds or extracts with recognized functionality may be included.

The term "food" in the present invention refers to meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, nutritional supplements, functional foods, food additives, health functional foods, and health foods, and include all foods in the conventional sense.

The above-mentioned health food refers to food that has a more active health maintenance or promotion effect compared to general food, and health supplement food refers to food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement are used interchangeably.

The term "health functional food" of the present invention is the same term as food for special health use (FoSHU), and refers to food with high medical and medical effects that has been processed to efficiently exhibit bioregulatory functions in addition to supplying nutrients. it means. Here, “function” means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The food of the present invention can be manufactured by a method commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various dosage forms and, unlike general drugs, has the advantage of not having side effects that may occur when taking the drug for a long period of time as it is made from food as a raw material, and is excellent in portability, making the composition of the present invention Food can be consumed as a supplement to prevent, suppress, or improve vascular aging or cardiovascular disease.

Specifically, the health functional foods are foods made by adding Senna alata extract to food materials such as beverages, teas, spices, gum, and confectionery, or manufactured into pills, tablets, capsules, powders, suspensions, etc., and when consumed It means that it brings about a specific health effect, but unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food.

The food composition of the present invention is very useful because it can be consumed on a daily basis and can be expected to prevent, suppress or improve vascular aging or cardiovascular disease.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated drinks, and electrolyte drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, It can be manufactured into foods such as cookies and noodles, and preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars. In addition, the food composition of the present invention may have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to Korea's 'Act on Health Functional Foods', or confectionery, tea, beverages, and special foods according to each food type according to the food code of Korea's 'Food Sanitation Act' (Ministry of Food and Drug Safety Notification 'Food Standards and Specifications'). It may be food for use, etc.

Additionally, the food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In terms of use, these food additives are divided into sweeteners, flavor enhancers, preservatives, emulsifiers, and flavorings.

The sweetener is used to impart an appropriate sweetness to food, and can be either natural or synthetic. For example, sweeteners such as neotame, lactitol, D-ribose, mannitol, D-maltitol, and sodium saccharin can be used.

The flavor enhancer is a food additive that enhances the taste or aroma of food, and both natural and synthetic agents can be used. For example, flavor enhancers may include disodium 5'-guanylate, L-glutamic acid, glycine, betaine, etc.

As the preservative, sodium metabisulfite, sulfurous anhydride, sorbic acid, benzoic acid, grapefruit seed extract, etc. may be used.

The emulsifier is a food additive that homogeneously mixes or maintains two or more immiscible phases such as water and oil, and may include sodium gluconate, glycerin fatty acid ester, lecithin, magnesium stearate, alginic acid, etc.

The flavoring agent is a food additive that gives a unique flavor to food or reinforces the original flavor of food lost during the manufacturing process. Ethyl vanillin, ethyl octanoate, linalyl acetate, allyl caproate, paramethylacetophenone, etc. may be used. .

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition. The bioactive substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine. Minerals include calcium preparations such as calcium citrate and stearic acid. Magnesium preparations such as magnesium, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose depending on the product type, and with respect to other food additives that may be included in the food composition of the present invention, each country's food code or You can refer to the Food Additives Code.

When using the Senna allata extract as a food additive, the Senna allata extract can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use.

In another aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating cardiovascular disease containing Senna alata extract as an active ingredient.

The terms “extract”, “active ingredient”, “cardiovascular disease”, and “prevention” of the present invention are as described above.

The term “treatment” of the present invention refers to any action that improves, alleviates, or benefits cardiovascular disease by administering the pharmaceutical composition of the present invention to an individual.

In the present invention, the composition can inhibit aging of vascular cells by reducing the activity of senescence associated beta galactosidase (SA-β-gal), an aging marker, in aged vascular cells.

In the present invention, the composition inhibits aging of vascular cells and normalizes the function of vascular cells by reducing the expression of aging markers p16, p21, p53, and PAI-1 (plasminogen activator inhibitor-1) in aged vascular cells. You can. The p53, p16, and p21 are proteins that inhibit cell growth or cell division, and the PAI-1 protein increases inflammation, promotes aging, and promotes the formation of blood clots.

SA-β-gal, p16, p21, p53, and PAI-1 are known as aging markers, and the correlation between vascular aging and cardiovascular disease is widely known in the art. Therefore, the composition containing the Senna alata extract according to the present invention as an active ingredient inhibits SA-β-gal activity and reduces the expression of aging-related proteins (p16, p21, p53, and PAI-1), thereby producing excellent vascular cells. Since it has an anti-aging effect, it is clear that a composition containing Senna alata extract has an effect in preventing or treating cardiovascular diseases.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. Pharmaceutically acceptable carriers are well known in the art depending on the administration route or dosage form, and for specifics, each country's pharmacopoeia, including the 'Korean Pharmacopoeia', can be referred to. 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, Examples include, but are not limited to, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, carriers, excipients, and diluents that may be included in the composition of the present invention may be unnatural carriers, but are not limited thereto.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods. . In detail, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are made by adding at least one excipient to the compound, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It can be prepared by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. You can. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, Withepsol, Macrogol, Tween 61, cacao, laurin, glycerogelatin, etc. can be used. Regarding the specific formulation of pharmaceutical compositions, it is known in the art and can refer to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount that is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects. The effective dose level refers to the patient's health status, type and severity of the disease, It can be determined based on factors including the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the medical field. The dosage and frequency of administration do not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered through various routes to mammals such as rats, dogs, cats, cows, horses, pigs, and humans, and humans may be preferable. All modes of administration are contemplated and may include, but are not limited to, oral, intravenous, intramuscular or subcutaneous injection.

The Senna allata extract according to the present invention inhibits senescence associated beta galactosidase (SA-β-gal) activity and reduces the expression of aging-related proteins (p16, p21, p53, and PAI-1), thereby excellently suppressing vascular cell aging. Because of its effectiveness, a composition containing Senna alata extract can not only be used to prevent or inhibit vascular aging, but can also be usefully used in the production of food and medicine for preventing, improving, or treating cardiovascular diseases.

Figure 1 is a graph showing the results of measuring the cytotoxicity of Senna alata extract.
Figure 2 is a micrograph confirming the cellular aging inhibition effect of Senna alata extract through SA-β-gal activity staining.
Figure 3 is a photograph of Western blotting results showing the expression levels of p16, p21, p53, and plasminogen activator inhibitor-1 (PAI-1) proteins in aged vascular endothelial cells treated with Senna alata extract.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Senna Alata ( Senna alata ) Extract preparation

Senna alata whole plant (i.e., the whole plant) was dried and then ground to a certain size. When using an automatic extractor (DIONEX Corporation, ASE300 ACCELERATED SOLVENT EXTRACTOR), 200 ml of methanol was added to 30 to 40 g of pulverized Senna alata, extracted for 20 minutes at 50°C and 1500 psi, and extracted with a sonicator (BRANSON Ultrasonics corporation, Ultrasonic). When a cleaner) was used, 1.5L of methanol was added to 200g of pulverized Senna alata and extraction was performed at 50°C for 3 days. After filtering the obtained extract, methanol was evaporated and concentrated to prepare a Senna alata methanol extract.

Experimental Example 1: Human umbrical vein endothelial cell (HUVEC) culture

HUVEC cells were distributed on a 100 cm plate using EBM-2 growth medium and cultured in a 5% carbon dioxide incubator at 37°C. When the cells grew to about 80% of the plate (1.3 x 10 ⁶ cells), the cells were separated by treatment with Trypsin-EDTA (0.25%) and passaged.

Experimental Example 2: Cytotoxicity measurement

To measure the effect of the Senna alata extract according to the present invention on cell survival, 0, 12.5, 25, 50, 100, or 200 μg/ml of Senna alata (SA) extract was added to cultured vascular endothelial cells. And cell viability was measured 48 hours later.

Specifically, vascular endothelial cells were distributed at ¹ The SA extract was treated and cultured at the above concentration. Based on 200uL medium, 10uL of D-Plus™ CCK cell viability assay kit solution was dispensed and the absorbance was measured at 450nm 1 hour later to check for cytotoxicity using CCK.

As a result, as shown in Figure 1A, it was confirmed that the SA extract increased cell viability. However, it was confirmed that toxicity occurred at a high concentration of 200ug/mL.

To confirm whether the SA extract is toxic to cells induced by senescence, vascular endothelial cells were treated with H ₂ O ₂ to induce senescence by oxidative stress. Specifically, 0, 12.5, 25, or 50 ug/mL of SA extract was treated with vascular endothelial cells, and 24 hours later, 100 ug/mL of H ₂ O ₂ was treated. After 72 hours, cell viability was confirmed.

Specifically, vascular endothelial cells were distributed at ¹ The SA extract is pretreated for 24 hours, then treated with H ₂ O ₂ for 72 hours and cultured. Based on 200uL medium, 10uL of D-Plus™ CCK cell viability assay kit solution was dispensed and the absorbance was measured at 450nm 1 hour later to check for cytotoxicity using CCK.

As a result, as shown in Figure 1B, in the group treated with H ₂ O ₂ , cytotoxicity appeared and cell survival rate decreased, but in the group treated with SA extract, cytotoxicity decreased and cell survival rate increased. It was confirmed that this was the case.

Experimental Example 3: Confirmation of cell aging inhibition effect of Senna alata extract through SA-β-gal activity staining

When aging was induced in vascular endothelial cells due to oxidative stress, the anti-aging effect of SA extract was observed using senescence associated beta galactosidase (SA-β-gal) staining to confirm the inhibitory effect of SA extract. The cytoplasm of aged cells is stained blue with SA-β-gal staining.

Specifically, vascular endothelial cells were distributed at ¹ Vascular endothelial cells were treated with 0, 12.5, 25, or 50 ug/mL of SA extract, and 24 hours later, they were treated with 100 ug/mL of H ₂ O ₂ . After 72 hours, cells were washed with 1x DPBS and fixed with 1x Fix solution (RT, 10min). After washing, 1x staining solution was added, sealed with parafilm, and reacted at 37°C overnight. After the reaction was completed, it was washed and the blue-stained cells were observed under a microscope.

As a result, as shown in Figure 2, blue SA-β staining was found to increase in the group treated with H ₂ O ₂ , and when treated with SA extract, the number of blue-stained cells was observed to decrease depending on the concentration of the SA extract. It has been done. Based on this, it was confirmed that the Senna alata extract according to the present invention can inhibit vascular aging and prevent or improve cardiovascular diseases caused by vascular aging.

Experimental Example 4: Confirmation of the effect of Senna alata extract on protein expression of aging-related genes

When aging is induced, protein expression of p16, p21, p53, and plasminogen activator inhibitor-1 (PAI-1) increases. Accordingly, we sought to determine the effect of SA extract on the expression of the aging-related proteins.

Specifically, vascular endothelial cells were distributed at ¹ Vascular endothelial cells were treated with 0, 12.5, 25, or 50 ug/mL of SA extract, and 24 hours later, they were treated with 100 ug/mL of H ₂ O ₂ . After 72 hours, proteins were extracted and Western blotting was performed to observe changes in aging-related protein expression. The cells were washed with DPBS, and 50-100uL of lysis buffer was added (the amount of lysis buffer is determined depending on the percentage of cells filled and cell state). Cell lysate was obtained by continuously vortexing on ice for 30 minutes. The supernatant was recovered by centrifugation at 13000 rpm at 4°C for 15 minutes. The amount of protein in the sample was quantified using the bradford method (bradford reagent, sigma). The proteins of the obtained samples were separated using a 10-15% acrylamide gel and transferred to a PVDF membrane. p16, p21, p53, and PAI-1 were attached as primary antibodies to the membrane to which the protein had been transferred, and anti-mouse was attached as a secondary antibody for antibody reaction. The protein expression level of the reacted PVDF membrane was confirmed using ECL™ Select Western Blotting Detection Reagent (Amersham, RPN2235).

As a result, as shown in Figure 3, in the group treated with H ₂ O ₂ , p16, p21, and PAI-1 protein expression increased due to cell aging, and in the group treated with SA extract, p16, p16, and PAI-1 protein expression increased in a SA extract concentration-dependent manner. p21, p53, and PAI-1 protein expression was decreased. Based on this, it was confirmed that the Senna alata extract according to the present invention can inhibit vascular aging and prevent or improve cardiovascular diseases caused by vascular aging.

Claims (13)

Prevention or inhibition of vascular aging caused by aging of vascular endothelial cells containing Senna alata methanol extract as an active ingredient; Or a food composition for preventing or improving cardiovascular disease caused by aging of vascular endothelial cells. delete delete According to paragraph 1,
The food composition, wherein the cardiovascular disease is selected from the group consisting of arteriosclerosis, hyperlipidemia, hypertension, angina, myocardial infarction, stroke, cardiac arrhythmia, heart failure, endocarditis, ischemic cerebrovascular disease, and ischemic heart disease. According to paragraph 1,
The composition is a food composition that reduces the activity of Senescence associated beta galactosidase (SA-β-gal) in aged vascular cells. According to paragraph 1,
The composition is a food composition that reduces the expression of p16, p21, p53, and PAI-1 (plasminogen activator inhibitor-1). Preventing or inhibiting vascular aging caused by vascular endothelial cell aging, comprising the food composition according to any one of claims 1 and 4 to 6; Or a health functional food for preventing or improving cardiovascular disease caused by aging of vascular endothelial cells. A pharmaceutical composition for preventing or treating cardiovascular disease caused by aging vascular endothelial cells, comprising a methanol extract of Senna alata as an active ingredient. delete delete According to clause 8,
A pharmaceutical composition wherein the cardiovascular disease is selected from the group consisting of arteriosclerosis, hyperlipidemia, hypertension, angina, myocardial infarction, and ischemic heart disease. According to clause 8,
The composition is a pharmaceutical composition that reduces the activity of senescence associated beta galactosidase (SA-β-gal) in aged vascular cells. According to clause 8,
The composition is a pharmaceutical composition that reduces the expression of p16, p21, p53, and PAI-1 (plasminogen activator inhibitor-1) in aged vascular cells.