Psychosaponin C as a composition for the treatment or prevention of age-related vascular diseases, including arteriosclerosis.

Abstract

The present invention relates to saikosaponin C as an agent for regulating vascular aging and associated arteriosclerosis, and provides a composition for treating, preventing or ameliorating aging vascular diseases, diseases caused by vascular aging, specifically arteriosclerosis, or symptoms caused by vascular aging, comprising saikosaponin C as an active ingredient. It is expected that the composition of the present invention can be used to treat, prevent or ameliorate arteriosclerosis as a complementary agent, alternative agent or combination agent for existing arteriosclerosis treatment agents that lower LDL-C.

Description

[Technical Field]

[0001] The present invention relates to psychosaponin C as a composition for the treatment or prevention of age-related vascular diseases, including arteriosclerosis.

[0002] [Background technology]

[0003] The most fundamental functional changes in blood vessels associated with vascular aging are impaired vasodilation (distensibility) and arterial stiffness. In other words, the development of arteriosclerosis is closely related to vascular aging.

[0004] However, to date, the treatment of arteriosclerosis has mainly consisted of statin drugs, which lower bad cholesterol (low-density lipoprotein cholesterol, LDL-C) in the blood, and stent surgery. Non-statin drugs are used in combination with or as complements to statins, but both statins and non-statins exert their effects by lowering LDL-C, the bad cholesterol. While these arteriosclerosis treatments have the advantage of showing excellent therapeutic effects on arteriosclerosis by suppressing LDL-C production, the therapeutic effect is temporary, and LDL-C levels return to their original levels if medication is stopped for about a week, so they have the limitation of requiring continuous administration.

[0005] Therefore, the need to develop a new concept in arteriosclerosis treatment based on the suppression of vascular aging has emerged. Considering that arteriosclerosis is a disease caused by vascular aging, this invention strives to develop a therapeutic agent that can maintain the health of the blood vessels themselves by suppressing vascular aging and thereby suppress the onset of arteriosclerosis. We have confirmed that this can dramatically reduce the number of medication doses and can also serve as a fundamental treatment, thus completing this invention. Furthermore, as it is an aging-suppression-based therapeutic agent, it can be expected to have preventive effects in addition to therapeutic effects, and is thought to be even more suitable for the treatment paradigm of aging diseases such as arteriosclerosis.

[0006] [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors of this invention have confirmed that atherosclerotic plaque sites exhibit a significantly more pronounced aging phenomenon compared to other vascular sites, and have confirmed that a considerable number of the active substances derived as vascular aging inhibitors exhibit efficacy as atherosclerotic treatment agents, thus confirming that atherosclerosis is a disease caused by vascular aging.

[0008] Furthermore, the inventors have confirmed that psychosaponin C suppresses vascular aging caused by vascular endothelial cell senescence, and that it suppresses monocyte-endothelial cell adhesion and macrophage foam cell formation by the NLRP3 inflammasome, which play important roles in the early stages of arteriosclerosis. Based on this, the inventors have confirmed that psychosaponin C can be used as a novel concept arteriosclerosis treatment agent based on the suppression of vascular aging, and have completed the present invention.

[0009] [Means for solving the problem]

[0010] The object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of diseases caused by vascular aging, comprising psychosaponin C or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] Another object of the present invention is to provide a food composition for improving or preventing diseases caused by vascular aging, comprising saikosaponin C or a food-grade salt thereof as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of symptoms caused by vascular aging, comprising psychosaponin C or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] Another object of the present invention is to provide a food composition for improving or preventing symptoms caused by vascular aging, comprising psychosaponin C or a food-grade salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide uses for psychosaponin C or pharmaceutically acceptable salts thereof for the manufacture of pharmaceuticals for the treatment or prevention of diseases caused by vascular aging.

[0015] Another object of the present invention is to provide uses for psychosaponin C or a food-grade salt thereof for the production of foods for improving or preventing diseases caused by vascular aging.

[0016] Another object of the present invention is to provide uses for psychosaponin C or pharmaceutically acceptable salts thereof for the manufacture of pharmaceuticals for the treatment or prevention of symptoms caused by vascular aging.

[0017] Another object of the present invention is to provide uses for psychosaponin C or a food-safe salt thereof for the production of foods for improving or preventing symptoms caused by vascular aging.

[0018] Another object of the present invention is to provide a method for treating or preventing diseases or symptoms caused by vascular aging, comprising the step of administering psychosaponin C or a pharmaceutically acceptable salt thereof to an individual.

[0019] [Effects of the Invention]

[0020] The present invention relates to the use of psychosaponin C, which exhibits simultaneous therapeutic effects against vascular aging and the resulting arteriosclerosis. It provides a composition containing psychosaponin C as an active ingredient for treating, preventing, or improving diseases caused by vascular aging or arteriosclerosis, and can be used effectively to treat, prevent, or improve vascular aging and arteriosclerosis.

[0021] [Brief explanation of the drawing]

[0022] [Figure 1] It was evaluated by MTT assay whether psychodysinin C alleviates the growth inhibition of vascular endothelial cells (HUVEC, HPAEC) by high-concentration glucose corresponding to a high-fat diet.

[0023] [Figure 2] It was evaluated by trypan blue staining whether psychodysinin C alleviates the growth inhibition of vascular endothelial cells (HUVEC, HPAEC) by high-concentration glucose.

[0024] [Figure 3] It was evaluated by measuring the expression of SA-β-gal staining and vascular aging marker proteins (PAI-1, ICAM-1, VCAM-1) whether psychodysinin C alleviates the aging of vascular endothelial cells (HUVEC, HPAEC) by high-concentration glucose.

[0025] [Figure 4] It was evaluated by measuring the cell cycle analysis and the expression of G1-phase growth delay-inducing proteins (p21, p27, p16) whether psychodysinin C alleviates the growth delay in the G1 phase of vascular endothelial cells (HUVEC, HPAEC) by high-concentration glucose.

[0026] [Figure 5] It was evaluated by fluorescence analysis whether psychodysinin C alleviates the generation of reactive oxygen species in vascular endothelial cells (HUVEC, HPAEC) by high-concentration glucose.

[0027] [Figure 6] It was evaluated whether psychodysinin C alleviates the decrease in nitric oxide concentration, nitric oxide synthase activity, protein expression, and phosphorylation decrease in vascular endothelial cells (HUVEC, HPAEC) by high-concentration glucose.

[0028] [Figure 7] This study evaluated whether psychosaponin C mitigates the decrease in Sirt1 activity, protein expression, and phosphorylation in vascular endothelial cells (HUVECs, HPAECs) caused by high glucose concentrations.

[0029] [Figure 8] To evaluate the mechanism of action of psychosaponin C in mitigating vascular endothelial cell (HUVEC) aging caused by high glucose concentrations, which are associated with high-fat diets, samples were treated with nitric oxide synthase inhibitors and Sirt1 protein inhibitors. The roles of nitric oxide synthase and Sirt1 protein in the anti-vascular aging efficacy of psychosaponin C, as well as the interrelationships between these proteins, were assessed.

[0030] [Figure 9] This study evaluated whether psychosaponin C inhibits the adhesion of mononuclear cells to vascular endothelial cells (HUVEC, HCAEC) induced by the arteriosclerosis-inducing agent TNF-α, by analyzing the adhesion of mononuclear cells labeled with a fluorescent substance (calcein AM) to vascular endothelial cells (HUVEC, HCAEC).

[0031] [Figure 10] This study evaluated whether psychosaponin C suppresses the non-ideal proliferation and migration of vascular smooth muscle cells (AoSMC, CASMC) mediated by PDGF, which is involved in the progression of arteriosclerosis, using wound healing analysis and Transwell cell migration analysis.

[0032] [Figure 11] This study evaluated, using Oil Red O staining, whether psychosaponin C suppresses macrophagous foam cell formation caused by oxidized LDL (ox-LDL), a typical finding in early-stage arteriosclerosis.

[0033] [Figure 12]This study evaluated whether psychosaponin C inhibits NLRP3 inflammasome activity, a protein complex that promotes macrophagous foam cell formation, using IL-1β secretion and caspase-1 cleavage analysis.

[0034] [Modes for carrying out the invention]

[0035] The present invention will be described in more detail below.

[0036]

[0037] On the other hand, each description and embodiment disclosed in the present invention is applicable to other descriptions and embodiments relating to each. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the specific descriptions below.

[0038] Expressions such as "including" used herein should be understood as open-ended terms that may include other embodiments, unless otherwise specified in the phrase or sentence containing the expression.

[0039] The terms and words used in the description and claims of this invention should not be interpreted in a manner limited to their ordinary or dictionary meanings, but rather in a manner consistent with the technical idea of ​​this invention, based on the principle that inventors may define the concepts of terms as appropriate to best describe their own invention.

[0040]

[0041] In one embodiment for achieving the above objective, the present invention provides a pharmaceutical composition for the treatment or prevention of diseases caused by vascular aging, comprising psychosaponin C or a pharmaceutically acceptable salt thereof as an active ingredient.

[0042] In this invention, the term saikosaponin C refers to one of the main components of the medicinal plant "Bupleuri Radix" and is a triterpene-type saponin. The molecular weight of saikosaponin C is 927.12, and its chemical formula is C 48 H 78 O 17 It relates to and has the structure of chemical formula 1.

[0043] [Chemical formula 1]

[0044] TIFF0007837595000001.tif62163

[0045] The pharmaceutical composition for the treatment or prevention of diseases caused by vascular aging, comprising psychosaponin C or a pharmaceutically acceptable salt thereof according to the present invention, has the effect of suppressing vascular aging by inhibiting the aging of vascular endothelial cells, and inhibits mononuclear cell-vascular endothelial cell adhesion and macrophagous foam cell formation by the NLRP3 inflammasome, which play an important role in the early stages of arteriosclerosis development, and can be used as a novel concept arteriosclerosis treatment agent based on the inhibition of vascular aging.

[0046] In this invention, the term "disease due to vascular aging" refers to a vascular disease induced by the aging of vascular endothelial cells, including arteriosclerosis, and more specifically, arteriosclerosis caused by the aging of vascular endothelial cells.

[0047] In the present invention, a composition containing psychosaponin C or a pharmaceutically acceptable salt thereof can suppress monocyte-endothelial cell adhesion, NLRP3 inflammasome activity, macrophage foam cell formation, or macrophage foam cell formation by the NLRP3 inflammasome, thereby treating or preventing arteriosclerosis, a disease caused by vascular aging.

[0048] In the present invention, the pharmaceutical composition contains saicosaponin C or a pharmaceutically acceptable salt thereof as an active ingredient, and may further contain a pharmaceutically acceptable carrier, and may be formulated by conventional methods in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, and sterile injection solutions.

[0049] As used in this invention, the term "pharmaceutically acceptable salt" means all salts of the compound that possess the desired biological and / or physiological activity and exhibit minimal undesirable toxicological effects. This means salts produced by conventional methods in the art, methods of which are known to those skilled in the art.

[0050] In the present invention, the pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry, for example, inorganic ion salts produced from calcium, potassium, sodium, and magnesium; inorganic salts produced from hydrochloric acid, nitric acid, phosphoric acid, bromate, iodic acid, perchloric acid, tartaric acid, and sulfuric acid; acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, and gluc. It may be any one selected from the group consisting of organic acid salts produced from ronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc., sulfonates produced from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid, amino acid salts produced from glycine, arginine, lysine, etc., and amine salts produced from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but is not limited thereto.

[0051] In the present invention, the pharmaceutical composition may contain a pharmaceutically acceptable carrier or additive. In the present invention, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what is appropriate for the target of application (formulation). The "carrier" is defined as a compound that facilitates the addition of a compound into cells or tissues.

[0052] The aforementioned pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the industry, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0053] Furthermore, the pharmaceutical composition of the present invention may contain fillers, bulking agents, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

[0054] The pharmaceutical composition of the present invention may be manufactured in the form of a liquid, suspension, powder, granules, tablet, capsule, pill, or extract.

[0055] The compositions of the present invention can be administered orally or parenterally (for example, topically or by intravenous, subcutaneous, or intraperitoneal injection).

[0056] In the present invention, the term "oral administration" refers to a method of administering a drug by mouth to improve pathological symptoms (periodontal disease), and in the present invention, the term "parenteral administration" refers to a method of administration other than oral administration, such as subcutaneous, intramuscular, intravenous, or intraperitoneal administration using a tube.

[0057] Solid preparations for oral administration include powders, granules, tablets, capsules, soft capsules, and pills. Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, syrups, and aerosols, but in addition to commonly used simple diluents such as water and liquid paraffin, they may also contain various excipients, such as humectants, sweeteners, fragrances, and preservatives.

[0058] Preparations for parenteral administration can be formulated and used in the form of topical preparations such as aqueous solutions, liquids, non-aqueous solvents, suspensions, emulsions, eye drops, eye ointments, syrups, suppositories, and aerosols, as well as sterile injectable preparations, each sterilized by conventional methods. Preferably, pharmaceutical compositions such as creams, gels, patches, sprays, ointments, plasters, lotions, liniments, eye ointments, eye drops, poultices, or compresses can be manufactured and used, but are not limited thereto. Compositions for topical administration may be anhydrous or aqueous depending on the clinical formulation. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[0059] The fillers, bulking agents, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives according to the present invention may be present in an amount of 0.1 to 99.9% by weight relative to the composition, and more specifically, may be present in an amount of 0.1 to 50% by weight, but is not limited thereto.

[0060] The pharmaceutical composition for the treatment or prevention of diseases caused by vascular aging, comprising psychosaponin C or a pharmaceutically acceptable salt thereof, of the present invention can be administered in the usual manner via oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intravascular routes, and may be administered orally.

[0061] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined by factors including the individual's species and severity, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, drugs used concurrently, and other factors well known in the medical field. For example, it may be administered once or several times a day in doses of 0.001 mg / kg to 5 g / kg, and this administration can be in a single dose or in multiple divided doses per day. The pharmaceutical composition of the present invention may also contain 0.001 to 50% by weight of psychosaponin C or a pharmaceutically acceptable salt thereof based on the total weight.

[0062] The pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar pharmacological effects as psychosaponin C or a pharmaceutically acceptable salt thereof, and can be administered in combination with existing vascular aging inhibitors or arteriosclerosis treatment agents.

[0063]

[0064] In another embodiment, the present invention provides a food composition for improving or preventing diseases caused by vascular aging, comprising saikosaponin C or a food-grade salt thereof as an active ingredient.

[0065] In this invention, the term "psychosaponin C" is explained as described above.

[0066] In this invention, the term "disease due to vascular aging" refers to a vascular disease induced by the aging of vascular endothelial cells, including arteriosclerosis, and more specifically, arteriosclerosis caused by the aging of vascular endothelial cells.

[0067] The food composition of the present invention may contain acceptable food additives, and may further contain suitable carriers, excipients, and diluents commonly used in the manufacture of food.

[0068] The food composition of the present invention may be in the form of pills, powders, granules, infusions, tablets, capsules, or liquids, and there are no particular limitations on the types of foods to which the saikosaponin C of the present invention or a food-grade salt thereof can be added. Examples of foods to which the substance can be added include meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen and other noodle products, gums, dairy products including ice cream, various soups, drinking water, tea, beverages, alcoholic beverages, and vitamin complexes.

[0069] The aforementioned food composition may contain other components besides saikosaponin C or a food-grade salt thereof, and the type of these components is not particularly limited. For example, like ordinary foods, it may contain, but is not limited to, various herbal extracts, food-grade food additives, or natural carbohydrates as additional components.

[0070] In the present invention, the term "food additive" means a component that can be added to food as an auxiliary, and is added to manufacture food in each dosage form, and can be appropriately selected and used by those skilled in the art. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages, but these examples do not limit the types of food additives in the present invention.

[0071] Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to those mentioned above, natural flavorings (such as thaumatin), stevia extracts (such as rebaudioside A and glycyrrhizin), and synthetic flavorings (such as saccharin and aspartame) can be advantageously used.

[0072] The food composition of the present invention may include health functional foods. In the present invention, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials and ingredients that have functional properties useful to the human body. Here, "functional" means obtaining effects useful for health purposes, such as regulating nutrients or physiological effects, on the structure and function of the human body. The health functional foods of the present invention can be manufactured by methods commonly used in the industry, and raw materials and ingredients commonly added in the industry can be added during the manufacturing process. Furthermore, unlike general pharmaceuticals, since it uses food as a raw material, it has the advantage of not having side effects that may occur with long-term use of pharmaceuticals, and it can be easily carried.

[0073] The amount of active ingredients mixed may be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment). Generally, during the manufacture of food, the active ingredients of the present invention may be added to the raw material composition in an amount of 0.01 to 50% by weight, preferably 0.1 to 10% by weight, but is not limited thereto. However, in the case of long-term intake for health and hygiene purposes or for health regulation purposes, the above amount may be used in amounts below the above range.

[0074]

[0075] In another embodiment, the present invention provides a pharmaceutical composition for the treatment or prevention of symptoms caused by vascular aging, comprising psychosaponin C or a pharmaceutically acceptable salt thereof as an active ingredient.

[0076] In this invention, the terms "psychosaponin C," "pharmaceutically acceptable salt," and "pharmaceutically acceptable composition" are explained above.

[0077] The symptoms resulting from the aforementioned vascular aging may be arteriosclerosis.

[0078] Furthermore, the symptoms due to vascular aging or arteriosclerosis may be one or more selected from the group consisting of: inhibition of vascular endothelial cell growth, increased vascular endothelial cell aging, increased production of reactive oxygen species by vascular endothelial cells, decreased nitric oxide concentration in vascular endothelial cells, decreased expression or activity of nitric oxide synthase, decreased expression or activity of Sirt1 protein, increased adhesion between mononuclear cells and vascular endothelial cells, increased NLRP3 inflammasome activity, increased macrophagous foam cell formation, and increased macrophagous foam cell formation by the NLRP3 inflammasome.

[0079] The pharmaceutical composition of the present invention, which contains psychosaponin C or a pharmaceutically acceptable salt thereof as an active ingredient, has the effect of treating or preventing the symptoms caused by vascular aging as described above.

[0080]

[0081] In another embodiment, the present invention provides a food composition for improving or preventing symptoms of vascular aging, comprising psychosaponin C or a food-grade salt thereof as an active ingredient.

[0082] In this invention, the terms "psychosaponin C," "food-grade acceptable salt," and "food composition" are explained as described above.

[0083] The aforementioned symptoms due to vascular aging may be indicative of arteriosclerosis.

[0084] Furthermore, the symptoms due to vascular aging or arteriosclerosis may be one or more selected from the group consisting of: inhibition of vascular endothelial cell growth, increased vascular endothelial cell aging, increased production of reactive oxygen species by vascular endothelial cells, decreased nitric oxide concentration in vascular endothelial cells, decreased expression or activity of nitric oxide synthase, decreased expression or activity of Sirt1 protein, increased adhesion between mononuclear cells and vascular endothelial cells, increased NLRP3 inflammasome activity, increased macrophagous foam cell formation, and increased macrophagous foam cell formation by the NLRP3 inflammasome.

[0085] The food composition of the present invention, which contains psychosaponin C or a food-grade salt thereof as an active ingredient, has the effect of improving or preventing the symptoms caused by vascular aging as described above.

[0086]

[0087] In another embodiment, the present invention provides uses for psychosaponin C or pharmaceutically acceptable salts thereof for the manufacture of pharmaceuticals for the treatment or prevention of diseases caused by vascular aging.

[0088] In another embodiment, the present invention provides uses for psychosaponin C or a food-safe salt thereof for the production of foods for improving or preventing diseases caused by vascular aging.

[0089] In another embodiment, the present invention provides uses for psychosaponin C or pharmaceutically acceptable salts thereof for the manufacture of pharmaceuticals for the treatment or prevention of symptoms caused by vascular aging.

[0090] In another embodiment, the present invention provides uses for psychosaponin C or a food-safe salt thereof for the production of foods for improving or preventing symptoms of vascular aging.

[0091] In another embodiment, the present invention provides a method for treating or preventing diseases or symptoms caused by vascular aging, comprising the step of administering psychosaponin C or a pharmaceutically acceptable salt thereof to an individual.

[0092] In this invention, the terms "psychosaponin C," "pharmaceutically acceptable salt," "food-acceptable salt," "diseases due to vascular aging," "symptoms due to vascular aging," "pharmaceutical composition," and "food composition" are explained as described above.

[0093] In the present invention, the term "individual" means any animal, including humans, that has developed a disease or symptoms due to vascular aging. This includes mammals, birds, etc., including cattle, pigs, sheep, chickens, dogs, and humans, and is not limited to individuals in which the disease or symptoms due to vascular aging are suppressed by the psychosaponin C of the present invention or a pharmaceutically acceptable salt thereof, thereby treating the disease or symptoms due to vascular aging.

[0094] [Examples]

[0095] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, so that they can be easily implemented by a person with ordinary skill in the art to which the present invention pertains. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0096]

[0097] Example 1. Preparation of Saikosaponin C

[0098] Saikosaponin C (SSc) is one of the main components of the medicinal plant Bupleuri Radix and belongs to the triterpene saponin group. Saikosaponin C has a molecular weight of 927.12 and its chemical formula is C 48 H 78 O 17 The substance in question was used in the following experiments at concentrations of 5 μM and 20 μM, and was purchased from Abcam (Cambridge, MA, USA).

[0099]

[0100] [Example of experiment]

[0101] Experimental Example 1. Mitigation effect of psychosaponin C on the inhibition of vascular endothelial cell growth induced by high glucose concentration (Substance Regeneration Analysis - MTT assay)

[0102] A high-concentration glucose (HG) solution of 33 mM, which is six times the standard glucose concentration of 5.5 mM in vascular endothelial cell culture medium, was used to construct a cellular-level high-fat diet evaluation system.

[0103] Specifically, two types of vascular endothelial cells, HUVECs (human umbilical vein endothelial cells) and HPAECs (human pulmonary artery endothelial cells), were pre-treated with psychosaponin C (SSc) at concentrations of 5 μM and 20 μM for 2 hours, and then treated with high-concentration glucose at a concentration of 33 mM for 72 hours. Subsequently, cell growth was analyzed by the MTT assay, which measures the mitochondrial substance regeneration capacity of living cells.

[0104] Figure 1 shows the results of an MTT assay evaluating whether psychosaponin C mitigates the inhibition of vascular endothelial cell (HUVEC, HPAEC) growth caused by high glucose concentrations associated with a high-fat diet. Vascular endothelial cell growth was evaluated by measuring the absorbance of MTT formazan at a wavelength of 590 nm.

[0105] As a result, high-concentration glucose treatment inhibited the growth of HUVECs and HPAECs. However, in HUVECs and HPAECs pretreated with psychosaponin C, the inhibition of cell growth by high-concentration glucose was mitigated, and it was confirmed that the mitigating effect of cell growth inhibition increased as the concentration of psychosaponin C increased.

[0106]

[0107] Experimental Example 2. Mitigation effect of psychosaponin C on the inhibition of vascular endothelial cell growth induced by high glucose concentration (cell count analysis - trypan blue staining).

[0108] A cellular-level high-fat diet evaluation system was constructed, identical to that used in Experimental Example 1.

[0109] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pre-treated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with high-concentration glucose at a concentration of 33 mM for 1 week. The number of living cells was then analyzed by trypan blue staining. The number of cells in the control group (none) was set to "100," and the relative values ​​are shown for comparison.

[0110] Figure 2 shows the results of an evaluation using trypan blue staining to determine whether psychosaponin C mitigates the inhibition of vascular endothelial cell (HUVEC, HPAEC) growth caused by high glucose concentrations associated with a high-fat diet. Vascular endothelial cell growth was evaluated by observing the cell number using trypan blue staining.

[0111] As a result, high-concentration glucose treatment inhibited the growth of HUVECs and HPAECs. However, in HUVECs and HPAECs pretreated with psychosaponin C, the inhibition of cell growth by high-concentration glucose was mitigated, and it was confirmed that the mitigating effect of cell growth inhibition increased as the concentration of psychosaponin C increased.

[0112]

[0113] Experimental Example 3. Analysis of the effect of psychosaponin C on mitigating vascular endothelial cell aging induced by high glucose concentrations (SA-β-gal staining and vascular aging-labeled protein expression analysis).

[0114] A cellular-level high-fat diet evaluation system was constructed, identical to that used in Experimental Example 1.

[0115] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pretreated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, respectively. Then, they were treated with high-concentration glucose at a concentration of 33 mM for 72 hours (for SA-β-gal staining) or 48 hours (for vascular senescence marker protein analysis). Subsequently, the degree of cellular senescence was measured by SA-β-gal staining, and the presence or absence of senescence was determined by the specific gravity of cells stained blue (SA-β-gal positive) among the total cells. Furthermore, the expression of vascular senescence marker proteins such as PAI-1, ICAM-1, and VCAM-1 was analyzed.

[0116] Figure 3 shows the results of evaluating whether psychosaponin C mitigates senescence of vascular endothelial cells (HUVECs, HPAECs) caused by high glucose concentrations associated with a high-fat diet, using SA-β-gal (senescence-associated β-galactosidase) staining and Western blotting. Specifically, senescence of vascular endothelial cells was evaluated by SA-β-gal staining, which measures the activity of β-galactosidase, an enzyme present in intracellular organelles called lysosomes (Figure 3(c), Figure 3(d)), and this was shown in graph form (Figure 3(a), Figure 3(b)). Furthermore, Figures 3(e) and 3(f) show the results of confirming the expression of vascular senescence-labeling proteins such as PAI-1, ICAM-1, and VCAM-1 in two types of vascular endothelial cells (HUVECs, HPAECs) by Western blotting.

[0117] As a result, high-concentration glucose treatment increased the specific gravity (Figure 3(a), Figure 3(b), Figure 3(c), Figure 3(d)) and the expression of vascular aging-labeling proteins (Figure 3(e), Figure 3(f)) of cells stained blue (SA-β-gal positive) in HUVEC and HPAEC cells, confirming that high-concentration glucose induces vascular cell aging. However, in HUVEC and HPAEC cells pretreated with psychosaponin C, the expression of SA-β-gal positive cells, PAI-1, ICAM-1, and VCAM-1 decreased, confirming a significant reduction in vascular cell aging induced by high-concentration glucose. In particular, it was confirmed that the inhibitory effect on vascular cell aging increased with increasing psychosaponin C concentration.

[0118]

[0119] Experimental Example 4. Analysis of the effect of psychosaponin C on mitigating G1 growth retardation induced by high glucose concentrations (cell cycle analysis and cell cycle regulatory protein expression analysis)

[0120] A cellular-level high-fat diet evaluation system was constructed, identical to that used in Experimental Example 1.

[0121] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pretreated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, then treated with high-concentration glucose (33 mM) for 48 hours, and finally fixed with cold 70% ethanol. The DNA of the fixed vascular endothelial cells was stained with PI (propidium iodide, 50 μg / ml) solution, and the cell cycle was analyzed using a fluid cell analyzer. In addition, after treating with high-concentration glucose (33 mM) for 36 hours, the expression of cell cycle regulatory proteins such as p21, p27, and p16 was analyzed.

[0122] Figure 4 shows the results of an evaluation of whether psychosaponin C mitigates the G1 phase growth arrest of vascular endothelial cells (HUVECs, HPAECs) caused by high glucose concentrations associated with a high-fat diet, using cell cycle analysis and measurement of the expression of G1 phase growth arrest-inducing proteins (p21, p27, p16). Since cellular senescence is closely related to G1 phase growth arrest in the cell cycle, the cell cycle was evaluated using a fluid cell analyzer (Figure 4(a), Figure 4(b)). In addition, the expression of the G1 phase growth arrest-inducing proteins p21, p27, and p16 was measured by Western blotting (Figure 4(c), Figure 4(d)).

[0123] As a result, we confirmed that high-concentration glucose treatment delayed G1 phase growth in the cell cycle and increased the expression of p21, p27, and p16 proteins in HUVEC and HPAEC cells. However, in HUVEC and HPAEC cells pretreated with psychosaponin C, the high-concentration glucose-induced G1 phase growth delay and the increase in p21, p27, and p16 protein expression were suppressed, and we confirmed that the inhibitory effect on G1 phase growth delay and the inhibitory effect on the increase in G1 phase growth delay-induced protein expression increased with increasing psychosaponin C concentration. G1 phase growth delay is a phenomenon observed in the cellular senescence process, and these results support the anti-vascular aging efficacy of psychosaponin C.

[0124]

[0125] Experimental Example 5. Analysis of the effect of psychosaponin C on mitigating reactive oxygen species generation induced by high-concentration glucose (fluorescence imaging analysis).

[0126] A cellular-level high-fat diet evaluation system was constructed, identical to that used in Experimental Example 1.

[0127] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pre-treated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with high-concentration glucose at a concentration of 33 mM for 24 hours. Subsequently, hydrogen peroxide (H2O2) and superoxide anion (O2) generated from the cytoplasm and mitochondria were analyzed. ·- The respective fluorescent probes were used to measure the values. In addition, samples treated with 100 μM hydrogen peroxide for 1 hour, followed by a medium change and further treatment for 23 hours were used as a positive control group for reactive oxygen species induction.

[0128] Different analytical reagents were used depending on the type of reactive oxygen species and its intracellular location of generation. Specifically, cytoplasmic hydrogen peroxide, intraplasmic superoxide anions, mitochondrial hydrogen peroxide, and mitochondrial superoxide anions were analyzed using H2DCFDA (2,7-dichlorodihydrofluorescein diacetate), DHE (dihydroethidium), DHR (dihydrorhodamine 123), and Mito-SOX reagents, respectively. H2DCFDA (1 μM, stained for 45 minutes), DHE (10 μM, stained for 60 minutes), DHR (20 μM, stained for 30 minutes), and Mito-SOX (5 μM, stained for 10 minutes) were used as reactive oxygen species-specific fluorescent probes for fluorescence image analysis.

[0129] Figure 5 shows the results of fluorescence imaging analysis to evaluate whether psychosaponin C mitigates the generation of reactive oxygen species (ROS) in vascular endothelial cells (HUVECs, HPAECs) caused by high glucose concentrations associated with a high-fat diet. Figure 5B shows the fluorescence imaging analysis results, and Figure 5A shows them in graph form.

[0130] As a result, it was confirmed that high-concentration glucose treatment increased the generation of various reactive oxygen species in HUVEC and HPAEC, including intracytoplasmic hydrogen peroxide, intracytoplasmic superoxide anions, mitochondrial hydrogen peroxide, and mitochondrial superoxide anions. However, in HUVEC and HPAEC pretreated with psychosaponin C, the generation of various reactive oxygen species induced by high-concentration glucose decreased, and the inhibitory effect on reactive oxygen species generation increased as the concentration of psychosaponin C increased. Since hydrogen peroxide and superoxide anions are representative reactive oxygen species, these results support the reactive oxygen species scavenging (ROS scavenging)-based anti-vascular aging efficacy of psychosaponin C.

[0131]

[0132] Experimental Example 6. Analysis of the effect of psychosaponin C on mitigating the reduction of NOS-NO signaling induced by high glucose concentrations (analysis of nitric oxide concentration, nitric oxide synthase activity, and protein expression).

[0133] A cellular-level high-fat diet evaluation system was constructed, identical to that used in Experimental Example 1.

[0134] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pre-treated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with high-concentration glucose at a concentration of 33 mM for 36 hours. Subsequently, nitric oxide (NO) concentration, endothelial nitric oxide synthase (eNOS) activity, protein expression, and the degree of phosphorylation were analyzed.

[0135] Figure 6 evaluates whether psychosaponin C mitigates the reduction in NOS-NO signaling in vascular endothelial cells (HUVECs, HPAECs) caused by high glucose concentrations associated with a high-fat diet. Nitric oxide generation was measured using a nitric oxide assay kit (Figure 6(a), Figure 6(b)). Specifically, nitrate (NO3) -) and nitrite (NO2 - ) values were analyzed by measuring them using nitrate reductase and Griess reagent. Nitric oxide synthase activity was measured using a NOS Activity assay kit (Figs. 6(c) and 6(d)), and the expression and phosphorylation (Ser114) of nitric oxide synthase were measured by Western blotting (Figs. 6(e) and 6(f)).

[0136] As a result, it was confirmed that the enzyme activity, protein expression, and phosphorylation of the Ser114 residue (phosphorylation, p-eNOS) of nitric oxide synthase (eNOS) decreased together with the nitric oxide (NO) value in HUVEC and HPAEC by high glucose treatment. However, the decrease in NOS-NO signal transduction by high glucose was alleviated in HUVEC and HPAEC pretreated with cycloastragenol C, and the alleviating effect of the decrease in NOS-NO signal transduction increased as the concentration of cycloastragenol C increased. Nitric oxide produced by nitric oxide synthase is a vasodilator that induces vasodilation, unlike reactive oxygen species, and maintains blood vessels in a healthy state. Such results support the anti-vascular aging efficacy of cycloastragenol C.

[0137]

[0138] [[ID=1))] Experimental Example 7. Analysis of the alleviating effect of cycloastragenol C on the decrease in Sirt1 activity / expression induced by high glucose (Sirt1 enzyme activity, protein expression, and phosphorylation analysis)

[0139] As in Experimental Example 1, a cell-level high-fat diet evaluation system was constructed.

[0140] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pretreated with cycloastragenol C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with high glucose at a concentration of 33 mM for 36 hours. Thereafter, the Sirt1 activity, protein expression, and degree of phosphorylation were analyzed.

[0141] Figure 7 shows the results of an evaluation of whether psychosaponin C mitigates Sirt1 activity, protein expression, and phosphorylation in vascular endothelial cells (HUVECs, HPAECs) induced by high glucose concentrations associated with a high-fat diet. Sirt1 activity was measured using a Sirt1 activity assay kit (Figure 7(a), Figure 7(b)), and Sirt1 protein expression and phosphorylation (Ser27, Ser47) were measured using Western blotting (Figure 7(c), Figure 7(d)).

[0142] As a result, high-concentration glucose treatment induced a decrease in Sirt1 enzyme activity, protein expression, and phosphorylation of the Ser27 / Ser47 residue in HUVEC and HPAEC. However, in HUVEC and HPAEC pretreated with psychosaponin C, the decrease in Sirt1 enzyme activity, protein expression, and phosphorylation caused by high-concentration glucose was mitigated, and the mitigating effect of the decrease in Sirt1 enzyme activity, protein expression, and phosphorylation increased with increasing concentration of psychosaponin C. Since Sirt1 protein expression is known to decrease during the aging process and exhibit an anti-vascular aging function that suppresses the aging of vascular cells, these results support the anti-vascular aging efficacy of psychosaponin C.

[0143]

[0144] Experimental Example 8. Analysis of the mechanism by which psychosaponin C mitigates vascular endothelial cell aging induced by high glucose concentrations (SA-β-gal staining, nitric oxide synthase activity, and Sirt1 enzyme activity analysis).

[0145] A cellular-level high-fat diet evaluation system was constructed, identical to that used in Experimental Example 1.

[0146] Specifically, vascular endothelial cells (HUVECs) were pretreated with psychosaponin C at a concentration of 20 μM for 2 hours, and then treated with high-concentration glucose at a concentration of 33 mM for 72 hours (for SA-β-gal staining) or 36 hours (for analysis of nitric oxide synthase activity and Sirt1 enzyme activity). Subsequently, SA-β-gal staining, nitric oxide synthase activity, and Sirt1 enzyme activity were analyzed. To determine whether nitric oxide synthase and Sirt1 protein are involved in the anti-vascular aging efficacy of psychosaponin C, and to analyze the relationship between nitric oxide synthase and Sirt1 protein, L-NAME (LN) is used as a nitric oxide synthase inhibitor. G We used Nitro arginine methyl ester (5 mM) and EX-527 (10 μM), an inhibitor of the Sirt1 protein.

[0147] Figure 8 shows the results of an evaluation of the relationship between nitric oxide synthase and Sirt1 protein, as well as whether nitric oxide synthase and Sirt1 protein are involved in the efficacy of psychosaponin C in mitigating vascular endothelial cell (HUVEC) aging caused by high glucose concentrations in a high-fat diet. Vascular endothelial cell aging was evaluated by intracellular SA-β-gal staining (Figure 8(a)), and the activity of nitric oxide synthase (Figure 8(b)) and Sirt1 protein (Figure 8(c)) were measured using the NOS activity assay kit and the Sirt1 activity assay kit, respectively.

[0148] As confirmed in Figure 3, high-concentration glucose treatment increased HUVEC senescence (SA-β-gal positive cells), and pretreatment with psychosaponin C mitigated HUVEC senescence. However, in HUVECs pretreated with L-NAME, an inhibitor of nitric oxide synthase, or EX-527, an inhibitor of Sirt1 protein, the anti-vascular aging effect of psychosaponin C was significantly inhibited. Furthermore, both L-NAME pretreatment and EX-527 pretreatment suppressed the effect of psychosaponin C in mitigating the decrease in nitric oxide synthase (NOS) activity, but L-NAME pretreatment did not suppress the effect of psychosaponin C in mitigating the decrease in Sirt1 protein activity.

[0149] These results suggest that Sirt1 protein activity and its subsequent nitric oxide synthase activity play a crucial role in the efficacy of psychosaponin C in suppressing vascular aging caused by high glucose concentrations.

[0150]

[0151] Experimental Example 9. Inhibitory effect of psychosaponin C on TNF-α-induced mononuclear cell-vascular endothelial cell adhesion during arteriosclerosis (fluorescence analysis and protein expression analysis).

[0152] From Experiment Example 3, we confirmed that psychosaponin C suppresses vascular endothelial cell aging. Since arteriosclerosis is a representative disease caused by vascular aging, we then tried to investigate whether psychosaponin C exhibits arteriosclerosis-inhibiting ability. The first arteriosclerosis analysis was performed by evaluating mononuclear cell-vascular endothelial cell adhesion, which plays an important role in the early stages of arteriosclerosis.

[0153] Specifically, two types of vascular endothelial cells, HUVEC (human umbilical vein endothelial cells) and HCAEC (human coronary artery endothelial cells), were pretreated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with TNF-α (20 ng / ml) for 8 hours. Subsequently, mononuclear cell-vascular endothelial cell adhesion and the expression of vascular endothelial cell adhesion proteins (ICAM-1, VCAM-1) were observed. As the mononuclear cell cell line, the human mononuclear cell line THP-1 was used, and the THP-1 cells were treated with the green fluorescent substance calcein AM (1 × 10⁻¹⁶). 6After leveling at 1 μg / ml per cell for 30 minutes, the solution was spread onto the two types of vascular endothelial cells mentioned above. Mononuclear cell-vascular endothelial cell adhesion was evaluated by imaging analysis using a confocal microscope, fluorescence analysis using a microplate reader, and Western blotting to assess the expression of adhesion proteins (ICAM-1, VCAM-1).

[0154] Figure 9 shows the results of an evaluation of whether psychosaponin C inhibits mononuclear cell-vascular endothelial cell adhesion induced by the arteriosclerosis-inducing agent TNF-α. Calcein AM fluorescence analysis using a confocal microscope and microplate reader, and the expression of cell adhesion proteins (ICAM-1, VCAM-1) were evaluated by Western blotting.

[0155] Figures 9(a) and 9(b) show the results of confocal microscopy analysis of mononuclear cell-endothelial cell adhesion in two types of vascular endothelial cells (HUVEC and HCAEC), and Figures 9(c) and 9(d) show the results of analysis of calcein AM fluorescence (excitation wavelength 488 nm, emission wavelength 517 nm) using a microplate reader in the same experiment. Figures 9(e) and 9(f) show the results of Western blotting confirmation of adhesion protein expression such as ICAM-1 and VCAM-1 in two types of vascular endothelial cells (HUVEC and HCAEC).

[0156] As a result, we confirmed that TNF-α treatment increased mononuclear cell-endothelial cell adhesion in both types of vascular endothelial cells (HUVEC and HCAEC). However, we confirmed that this adhesion was suppressed when pre-treated with psychosaponin C. Furthermore, we confirmed that psychosaponin C suppresses the expression of adhesion proteins on vascular endothelial cells involved in mononuclear cell-endothelial cell adhesion. Mononuclear cell-endothelial cell adhesion is considered to be an early stage of atherosclerosis development, and adhesion proteins such as ICAM-1 and VCAM-1 are also vascular aging-labeling proteins. Therefore, these results suggest that psychosaponin C has anti-vascular aging efficacy and, based on that, anti-atherosclerotic efficacy.

[0157]

[0158] Experimental Example 10. Analysis of the inhibitory effect of psychosaponin C on PDGF-induced vascular smooth muscle cell migration during arteriosclerosis (Wound healing ability and cell migration analysis)

[0159] As shown in Experimental Example 9 above, we aimed to determine whether psychosaponin C exhibits arteriosclerosis-inhibiting activity. The second arteriosclerosis analysis was performed by evaluating the non-ideal proliferation and migration of vascular smooth muscle cells, which are the main mechanisms that signal the progression to full-blown arteriosclerosis.

[0160] Specifically, two types of vascular smooth muscle cells, AoSMC (human aortic smooth muscle cells) and CASMC (human coronary artery smooth muscle cells), were pretreated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with platelet-derived growth factor (PDGF) PDGF-BB (20 ng / ml) for 24 hours. Subsequently, the proliferation and migration of vascular smooth muscle cells were observed. A wound healing assay was performed to observe the proliferation and migration of vascular smooth muscle cells, and a Transwell migration assay was performed to observe the migratory capacity of vascular smooth muscle cells.

[0161] Figure 10 shows the results of an evaluation of whether psychosaponin C inhibits the proliferation and migration of vascular smooth muscle cells induced by the arteriosclerosis-inducing agent PDGF-BB. Figures 10(a) and 10(b) show the results of evaluating the proliferation and migration of vascular smooth muscle cells (AoSMC and CASMC) in two types of vascular smooth muscle cells (AoSMC and CASMC) using scratch wound healing analysis. Figures 10(c) and 10(d) show the results of analyzing cell migration in the same cells using an 8.0 μm pore-sized Transwell migration chamber.

[0162] As a result, we confirmed that PDGF-BB treatment increased the proliferation and migration of vascular smooth muscle cells in both types of vascular smooth muscle cells (AoSMC and CASMC). Unlike in the previous experimental example 9, we confirmed that pretreatment with psychosaponin C did not suppress this proliferation and migration. These results suggest that the proliferation and migration of vascular smooth muscle cells are not involved in the anti-atherosclerotic efficacy of psychosaponin C.

[0163]

[0164] Experimental Example 11. Inhibitory effect of psychosaponin C on macrophageous foam cell formation during arteriosclerosis (imaging analysis and absorbance analysis)

[0165] As shown in Experimental Examples 9 and 10 above, psychosaponin C inhibited mononuclear cell-vascular endothelial cell adhesion mediated by TNF-α, but did not inhibit PDGF-mediated abnormal proliferation and migration of vascular smooth muscle cells. Therefore, we attempted to investigate whether it exhibits atherosclerosis-inhibiting ability using other atherosclerotic phenotypic analyses. The third atherosclerosis analysis was performed by observing macrophage foam cell formation, which plays an important role in the early stages of atherosclerosis, along with mononuclear cell-vascular endothelial cell adhesion. Macrophage foam cell formation is a stage in the progression to atherosclerosis that occurs between mononuclear cell-vascular endothelial cell adhesion and the non-ideal proliferation and migration of vascular smooth muscle cells.

[0166] Specifically, THP-1 cells, a mononuclear cell line, were treated with PMA (Phorbol 12-myristate 13-acetate) at a concentration of 150 nM for 48 hours to differentiate them into macrophages. The differentiated macrophages were pre-treated with psychosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with oxidized LDL (ox-LDL) (100 μg / ml), a foam cell inducer, for 24 hours. Subsequently, the formation of foam cells from macrophages was observed. Foam cell formation was evaluated by microscopic analysis after staining with Oil Red O, which adsorbs to lipid molecules, and by absorbance analysis using a microplate reader.

[0167] Figure 11 evaluates whether psychosaponin C inhibits macrophagocyte foam cell formation caused by oxidized LDL (ox-LDL), an arteriosclerosis inducer. Figure 11 shows the results of analyzing the degree of macrophagocyte foam cell formation after fixing cells in 10% formalin for 30 minutes and staining them with Oil Red O for 30 minutes. The nucleus was stained with hematoxylin for 1 minute. The degree of foam cell formation was analyzed by microscopic analysis of the degree of red staining with Oil Red O, as shown in Figure 11(a), or by measuring the absorbance at 540 nm using a microplate reader after eluting the Oil Red O stain with 100% isopropanol for 10 minutes, as shown in Figure 11(b).

[0168] As a result, we confirmed that oxidized LDL treatment caused macrophages to take in large amounts of lipids and convert into foam cells. However, we confirmed that pretreatment with psychosaponin C suppressed foam cell formation due to such lipid deposition. Since the conversion of macrophages to foam cells, along with mononuclear cell-vascular endothelial cell adhesion, is considered a typical pathological finding of early arteriosclerosis, these results suggest that psychosaponin C acts in the early stages of arteriosclerosis and has anti-arteriosclerotic efficacy.

[0169]

[0170] Experimental Example 12. Inhibitory effect of psychosaponin C on NLRP3 inflammasome activity during arteriosclerosis (ELISA analysis and protein expression analysis)

[0171] As shown in Experimental Examples 9, 10, and 11 above, we attempted to determine whether psychosaponin C exhibits arteriosclerosis-inhibiting activity. NLRP3 (NOD-like receptor protein-3), which plays a sensor role, forms a protein complex called the NLRP3 inflammasome together with ASC, which plays an adapter role, and caspase-1, which plays an effector role. In response to infection and cell damage, the NLRP3 inflammasome causes pyroptosis, a cell death associated with inflammation, through the secretion of the inflammatory cytokine IL-1β and activation of caspase-1. Above all, the NLRP3 inflammasome is known to promote macrophagous foam cell formation through IL-1β secretion and caspase-1 activation. Since Experimental Example 11 showed that psychosaponin C inhibits macrophagous foam cell formation by oxidized LDL, the fourth arteriosclerosis analysis was performed by evaluating the activity of the NLRP3 inflammasome, which promotes macrophagous foam cell formation.

[0172] Specifically, THP-1 cells, a mononuclear cell line, were treated with PMA at a concentration of 150 nM for 48 hours to differentiate them into macrophages. The differentiated macrophages were pre-treated with psychosaponin at a concentration of 20 μM for 2 hours, and then treated with monosodium urate crystal (MSU), an NLRP3 inflammasome inducer (activator), at a concentration of 200 μg / ml for 6 hours. Subsequently, IL-1β secretion and caspase-1 cleavage were analyzed. In addition, as a control group for comparing the efficacy of psychosaponin C, the NLRP3 inflammasome inhibitor MCC950 (1 μM) was pre-treated for 2 hours in the same manner as psychosaponin C.

[0173] Figure 12 evaluates whether psychosaponin C inhibits NLRP3 inflammasome activity, which promotes macrophagous foam cell formation observed in early arteriosclerosis. Figure 12(a) shows the results of analyzing NLRP3 inflammasome activity by measuring IL-1β secretion, an inflammatory cytokine, using solid-phase sandwich ELISA. Figure 12(b) shows the results of analyzing NLRP3 inflammasome activity by evaluating caspase-1 cleavage using Western blotting.

[0174] As a result, we confirmed that urate crystal (MSU) treatment increased IL-1β secretion and caspase-1 cleavage. However, we confirmed that pretreatment with psychosaponin C, similar to pretreatment with the NLRP3 inflammasome inhibitor MCC950, suppressed these IL-1β secretion and caspase-1 cleavage. Since caspase-1 cleavage and subsequent IL-1β secretion are necessary for NLRP3 inflammasome activation, this suppression of IL-1β secretion and caspase-1 cleavage means that psychosaponin C can suppress NLRP3 inflammasome activity. Furthermore, since activated NLRP3 inflammasome promotes foam cell formation, these results further demonstrate the anti-atherosclerotic efficacy of psychosaponin C in the early stages of atherosclerosis by suppressing macrophagocyte foam cell formation.

[0175]

[0176] In short, psychosaponin C suppressed vascular aging caused by vascular endothelial cell senescence and inhibited mononuclear cell-vascular endothelial cell adhesion and macrophagous foam cell formation by the NLRP3 inflammasome, which play important roles in the early stages of atherosclerosis. Therefore, psychosaponin C can be used as a vascular aging-inhibiting atherosclerosis treatment agent. Such a novel concept vascular aging-inhibiting atherosclerosis treatment agent can be used alone to independently demonstrate therapeutic effects for atherosclerotic diseases, or it can be used in combination with existing atherosclerosis treatment agents that lower LDL-C to enhance therapeutic effects.

[0177]

[0178] Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concepts of the present invention as defined in the attached claims also fall within the scope of the present invention.

Claims

1. A pharmaceutical composition for the treatment or prevention of arteriosclerosis, comprising psychosaponin C or a pharmaceutically acceptable salt thereof as an active ingredient, The treatment or prevention described above is a pharmaceutical composition comprising the suppression of monocyte-endothelial cell adhesion, NLRP3 inflammasome activity, macrophage foam cell formation, or macrophage foam cell formation by the NLRP3 inflammasome.

2. The pharmaceutical composition according to claim 1, wherein the arteriosclerosis is a disease or symptom caused by vascular aging.

3. The pharmaceutical composition according to claim 2, wherein the vascular aging is the aging of vascular endothelial cells.

4. A food composition for improving or preventing arteriosclerosis, comprising saikosaponin C or a food-grade salt thereof as an active ingredient, The improvement or prevention described above is a food composition comprising suppressing mononuclear cell-endothelial cell adhesion, NLRP3 inflammasome activity, macrophage foam cell formation, or macrophage foam cell formation by the NLRP3 inflammasome.

5. The food composition according to claim 4, wherein the arteriosclerosis is a disease or symptom caused by vascular aging.

6. The food composition according to claim 5, wherein the vascular aging is the aging of vascular endothelial cells.

7. The food composition according to claim 4, wherein the food composition is a health functional food.

8. The pharmaceutical composition according to claim 2, wherein the symptoms due to vascular aging are one or more selected from the group consisting of inhibition of vascular endothelial cell growth, increased vascular endothelial cell aging, increased production of reactive oxygen species by vascular endothelial cells, decreased nitric oxide concentration in vascular endothelial cells, decreased expression or activity of nitric oxide synthase, decreased expression or activity of Sirt1 protein, increased adhesion between mononuclear cells and vascular endothelial cells, increased NLRP3 inflammasome activity, increased macrophageous foam cell formation, and increased macrophageous foam cell formation by NLRP3 inflammasome.

9. The food composition according to claim 5, wherein the symptoms due to vascular aging are one or more selected from the group consisting of inhibition of vascular endothelial cell growth, increased vascular endothelial cell aging, increased reactive oxygen species generation by vascular endothelial cells, decreased nitric oxide concentration in vascular endothelial cells, decreased expression or activity of nitric oxide synthase, decreased expression or activity of Sirt1 protein, increased adhesion between mononuclear cells and vascular endothelial cells, increased NLRP3 inflammasome activity, increased macrophageous foam cell formation, and increased macrophageous foam cell formation by NLRP3 inflammasome.

Images