Saikosaponin C as a composition for treating or preventing aging-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 saikosaponin C as a composition for treating or preventing age-related vascular diseases, including arteriosclerosis.

[0002] [Background technology]

[0003] The most fundamental functional change in blood vessels associated with vascular aging is the impairment of vascular distensibility and arterial stiffness. In other words, the development of arteriosclerosis is closely related to vascular aging.

[0004] However, the treatment of arteriosclerosis to date has mainly consisted of stent surgery and statin drugs to lower the bad cholesterol (LDL-C) in the blood. Non-statin drugs are used in combination with statin drugs or as complementary drugs, and both statin and non-statin drugs work by lowering LDL-C, the bad cholesterol. These arteriosclerosis treatment drugs have the advantage of showing excellent therapeutic effects on arteriosclerosis by suppressing LDL-C production, but the therapeutic effect is temporary, and if the medication is stopped for about a week, the original LDL-C value will return, so there is a limit to the need to continue taking the medication.

[0005] Therefore, there is an increasing need to develop a new concept arteriosclerosis treatment agent based on the inhibition of vascular aging. Considering that arteriosclerosis is a disease caused by vascular aging, the present invention has been developed to develop a treatment agent that can maintain the blood vessels themselves healthy and inhibit the onset of arteriosclerosis by inhibiting vascular aging, and has confirmed that it can dramatically reduce the number of times of administration and can also be a fundamental treatment agent, leading to the completion of the present invention. In addition, since it is a treatment agent based on aging inhibition, it is expected to have preventive efficacy in addition to therapeutic efficacy, and is considered to be more suitable for the treatment paradigm of aging diseases such as arteriosclerosis.

[0006] Summary of the Invention [Problem to be solved by the invention]

[0007] The inventors of the present invention have confirmed that arteriosclerotic plaque sites show more significant aging phenomena than other blood vessel sites, and have confirmed that a significant number of effective substances derived as vascular aging inhibitors show efficacy as arteriosclerosis treatment agents, and have confirmed that arteriosclerosis is a disease caused by vascular aging.

[0008] In addition, the inventors have confirmed that Saikosaponin C inhibits vascular aging caused by vascular endothelial cell aging, and inhibits monocyte-endothelial cell adhesion and macrophage foam cell formation caused by NLRP3 inflammasome, which play important roles in the early stage of arteriosclerosis pathogenesis. They have confirmed that Saikosaponin C can be used as a new concept arteriosclerosis treatment agent based on inhibiting vascular aging, and have completed the present invention.

[0009] [Means for solving the problem]

[0010] An object of the present invention is to provide a pharmaceutical composition for treating or preventing diseases caused by vascular aging, which comprises saikosaponin C or a pharma- ceutically 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, which comprises saikosaponin C or a dietarily acceptable salt thereof as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition for treating or preventing symptoms caused by vascular aging, comprising saikosaponin C or a pharma- ceutically 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, which comprises saikosaponin C or a dietarily acceptable salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide a use of Saikosaponin C or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of a disease caused by vascular aging.

[0015] Another object of the present invention is to provide use of saikosaponin C or a food-based acceptable salt thereof for the production of a food for improving or preventing diseases caused by vascular aging.

[0016] Another object of the present invention is to provide a use of Saikosaponin C or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of symptoms due to vascular aging.

[0017] Another object of the present invention is to provide a use of saikosaponin C or a food-based acceptable salt thereof for producing a food 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 a disease caused by vascular aging or a symptom caused by vascular aging, comprising the step of administering Saikosaponin C or a pharma- ceutically acceptable salt thereof to an individual.

[0019] Effect of the Invention

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

[0021] [Brief description of the drawings]

[0022] [Figure 1] The MTT assay was used to evaluate whether Saikosaponin C alleviates the growth inhibition of vascular endothelial cells (HUVEC, HPAEC) caused by high-concentration glucose, which corresponds to a high-fat diet.

[0023] [Diagram 2] The ability of Saikosaponin C to alleviate the growth inhibition of vascular endothelial cells (HUVEC, HPAEC) caused by high concentration glucose was evaluated using trypan blue staining.

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

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

[0026] [Diagram 5] This study used fluorescence analysis to evaluate whether Saikosaponin C alleviates the generation of reactive oxygen species in vascular endothelial cells (HUVEC, HPAEC) caused by high concentrations of glucose.

[0027] [Figure 6] This study evaluated whether Saikosaponin C attenuates the high glucose-induced decrease in nitric oxide concentration, nitric oxide synthase activity, protein expression and phosphorylation in vascular endothelial cells (HUVEC, HPAEC).

[0028] [Figure 7] This study evaluated whether Saikosaponin C attenuates the high glucose-induced decrease in Sirt1 activity, protein expression, and phosphorylation in vascular endothelial cells (HUVEC, HPAEC).

[0029] [Figure 8] To evaluate the mechanism of action of Saikosaponin C in alleviating aging of vascular endothelial cells (HUVECs) caused by high glucose concentrations, which corresponds to a high-fat diet, we treated them with nitric oxide synthase inhibitors and Sirt1 protein inhibitors to evaluate the role of nitric oxide synthase and Sirt1 protein in the anti-vascular aging efficacy of Saikosaponin C, and the relationship between these proteins.

[0030] [Figure 9] Whether Saikosaponin C inhibits monocyte-vascular endothelial cell adhesion caused by TNF-α, an atherosclerosis-inducing agent, was evaluated by an adhesion analysis of monocytes labeled with a fluorescent substance (calcein AM) to vascular endothelial cells (HUVEC, HCAEC).

[0031] [Figure 10] We evaluated whether Saikosaponin C inhibits the non-ideal proliferation and migration of vascular smooth muscle cells (AoSMC, CASMC) caused by PDGF, which is involved in the progression of arteriosclerosis, using wound healing assay and transwell cell migration assay.

[0032] [Figure 11] Whether Saikosaponin C inhibits macrophage foam cell formation caused by oxidized LDL (ox-LDL), a typical finding of early-stage arteriosclerosis, was evaluated using Oil Red O staining.

[0033] [Figure 12]We assessed whether Saikosaponin C inhibits NLRP3 inflammasome activity, a protein complex that promotes macrophage foam cell formation, using IL-1β secretion and caspase-1 cleavage assays.

[0034] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0036]

[0037] Meanwhile, each description and embodiment disclosed in the present invention can be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific description below.

[0038] As used herein, terms such as "comprises" should be understood as open-ended terms that include the possibility of including other embodiments, unless otherwise indicated in the phrase or sentence in which the term appears.

[0039] The terms and words used in the description of the present invention and the claims should not be interpreted in a limited manner to their ordinary or dictionary meanings, but should be interpreted in a meaning and concept that corresponds to the technical idea of ​​the present invention, based on the principle that an inventor can appropriately define the concept of a term in order to best describe his own invention.

[0040]

[0041] In one aspect for achieving the above-mentioned object, the present invention provides a pharmaceutical composition for treating or preventing diseases caused by vascular aging, comprising saikosaponin C or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0042] In the present invention, the term saikosaponin C is one of the main components of the medicinal plant "Bupleuri Radix" and corresponds to triterpene saponins. The molecular weight of saikosaponin C is 927.12 and the chemical formula is C 48 H 78 O 17 and has the structure of Chemical Formula 1.

[0043] [Chemical formula 1]

[0044] TIFF2025508556000002.tif62163

[0045] The pharmaceutical composition for treating or preventing diseases caused by vascular aging, comprising Saikosaponin C or a pharma- ceutically acceptable salt thereof according to the present invention, has the effect of inhibiting vascular aging by inhibiting aging of vascular endothelial cells, and inhibits monocyte-vascular endothelial cell adhesion and macrophage foam cell formation by NLRP3 inflammasome, which play important roles in the early stage of the onset of arteriosclerosis, and can be used as a new concept arteriosclerosis treatment agent based on the inhibition of vascular aging.

[0046] In the present invention, the term "disease caused by vascular aging" refers to a vascular disease induced by aging of vascular endothelial cells, such as arteriosclerosis, and specifically may be arteriosclerosis caused by aging of vascular endothelial cells.

[0047] In the present invention, a composition containing Saikosaponin C or a pharma- ceutically acceptable salt thereof can inhibit monocyte-endothelial cell adhesion, NLRP3 inflammasome activity, macrophage foam cell formation, or macrophage foam cell formation by NLRP3 inflammasome, and can treat or prevent arteriosclerosis, a disease caused by vascular aging.

[0048] In the present invention, the pharmaceutical composition contains saikosaponin C or a pharma- ceutically acceptable salt thereof as an active ingredient, and may further contain a pharma- ceutically acceptable carrier, and may be formulated by conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, and sterile injectable solutions.

[0049] The term "pharmaceutically acceptable salt" as used herein means any salt that retains the desired biological and / or physiological activity of the compound and exhibits minimal undesired toxicological effects, and is prepared by conventional methods in the art, which are well known to those skilled in the art.

[0050] In the present invention, the pharma- ceutically acceptable salt refers to a salt commonly used in the pharmaceutical industry, and examples of such salts include inorganic ion salts prepared from calcium, potassium, sodium, magnesium, etc., inorganic acid salts prepared from hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid, sulfuric acid, etc., 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, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glycer ... The salt may be any one selected from the group consisting of organic acid salts prepared from sucrose, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc.; sulfonate salts prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; amino acid salts prepared from glycine, arginine, lysine, etc.; and amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but is not limited thereto.

[0051] In the present invention, the pharmaceutical composition may contain a pharma- ceutically acceptable carrier or additive. In the present invention, the term "pharma-ceutically acceptable" means that it does not suppress the activity of the active ingredient and does not have toxicity beyond the range that the subject of application (prescription) can handle. The "carrier" is defined as a compound that facilitates the addition of a compound to cells or tissues.

[0052] The pharma- ceutically acceptable carriers include those commonly used in the art, such as, but not limited to, 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 diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and other pharma- ceutically acceptable additives.

[0054] The pharmaceutical compositions of the present invention may be prepared in the form of a liquid, suspension, powder, granules, tablets, capsules, pills, or extract.

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

[0056] In the present invention, the term "oral administration" refers to a method of administering a drug for improving a pathological condition (periodontal disease) through the mouth, and in the present invention, the term "parenteral administration" refers to a method of administration subcutaneously, intramuscularly, intravenously, or intraperitoneally using a tube, excluding administration through the mouth.

[0057] Solid preparations for oral administration include powders, granules, tablets, capsules, soft capsules, pills, etc. Liquid preparations for oral administration include suspensions, liquids, emulsions, syrups, aerosols, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents.

[0058] For parenteral administration, the pharmaceutical compositions may be prepared and used in the form of external preparations such as sterilized aqueous solutions, liquids, non-aqueous solvents, suspensions, emulsions, eye drops, eye ointments, syrups, suppositories, aerosols, etc., and sterile injection preparations, each of which may be prepared by a conventional method. Preferably, the pharmaceutical compositions may be prepared and used in the form of creams, gels, patches, sprays, ointments, plasters, lotions, liniments, eye ointments, eye drops, poultices, or compresses, but are not limited thereto. The compositions for topical administration may be anhydrous or aqueous, depending on clinical prescriptions. For non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. For suppository bases, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. may be used.

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

[0060] The pharmaceutical composition for treating or preventing diseases caused by vascular aging, comprising Saikosaponin C or a pharma- ceutical acceptable salt thereof, of the present invention may be administered in a conventional manner via oral, rectal, intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intravascular routes, and specifically may be administered orally.

[0061] The pharmaceutical composition of the present invention is administered in a pharma- tically effective amount. The term "pharma-tically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level may be determined by factors including the type and severity of the individual, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. For example, it may be administered at a dose of 0.001 mg / kg to 5 g / kg once or several times a day, and this administration may be administered in a single dose or multiple doses per day. In addition, the pharmaceutical composition of the present invention may contain 0.001 to 50% by weight of saikosaponin C or a pharma- tically 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 in addition to saikosaponin C or a pharma- ceutically acceptable salt thereof, and can be administered in combination with existing agents for inhibiting vascular aging or treating arteriosclerosis.

[0063]

[0064] In another aspect, the present invention provides a food composition for improving or preventing diseases caused by vascular aging, which comprises saikosaponin C or a dietarily acceptable salt thereof as an active ingredient.

[0065] In the present invention, the explanation for the term "saikosaponin C" is as described above.

[0066] In the present invention, the term "disease caused by vascular aging" refers to a vascular disease induced by aging of vascular endothelial cells, such as arteriosclerosis, and specifically, may be arteriosclerosis caused by aging of vascular endothelial cells.

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

[0068] The food composition of the present invention may be in the form of pills, powders, granules, infusions, tablets, capsules, or liquids, and there is no particular limit to the types of foods to which the Saikosaponin C or its food-scientifically acceptable salt of the present invention can be added. Examples of foods to which the substance can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes.

[0069] The food composition may contain other ingredients in addition to Saikosaponin C or its nutrient-acceptable salt, and the types of ingredients are not particularly limited. For example, like ordinary foods, the food composition may contain various herbal extracts, nutrient-acceptable food supplements, natural carbohydrates, etc. as additional ingredients, but is not limited thereto.

[0070] In the present invention, the term "food supplement additive" refers to a component that can be added supplementarily to food, and is added to produce food of various dosage forms, and can be appropriately selected and used by those skilled in the art. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc., but the types of food supplement additives of the present invention are not limited by these examples.

[0071] Examples of the 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. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, etc.), stevia extracts (rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

[0072] The food composition of the present invention may include a functional health food. In the present invention, the term "functional health food" refers to a food manufactured and processed in the form of tablets, capsules, powder, granules, liquid, pills, etc., using raw materials or ingredients having useful functions for the human body. Here, functionality means obtaining useful effects for health purposes such as regulating nutrients or physiological effects on the structure and function of the human body. The functional health food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, unlike general medicines, since it uses food as a raw material, it has the advantage of being free of side effects that may occur when taking medicines for a long period of time, and it can be easily carried around.

[0073] The amount of the active ingredient to be mixed may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, when producing food, the active ingredient 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 the purpose of health and hygiene or health regulation, the amount may be used within the above range.

[0074]

[0075] In another aspect, the present invention provides a pharmaceutical composition for treating or preventing symptoms caused by vascular aging, comprising saikosaponin C or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0076] In the present invention, the terms "saikosaponin C," "pharmaceutical acceptable salt," and "pharmaceutical composition" are as described above.

[0077] The symptom caused by vascular aging may be arteriosclerosis.

[0078] In addition, the symptoms due to vascular aging or symptoms due to 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 in 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 monocyte-vascular endothelial cell adhesion, increased NLRP3 inflammasome activity, increased macrophage foam cell formation, and increased macrophage foam cell formation by NLRP3 inflammasome.

[0079] The pharmaceutical composition of the present invention containing saikosaponin C or a pharma- ceutically acceptable salt thereof as an active ingredient is effective in treating or preventing the above-mentioned symptoms caused by vascular aging.

[0080]

[0081] In another aspect, the present invention provides a food composition for improving or preventing symptoms caused by vascular aging, comprising saikosaponin C or a dietarily acceptable salt thereof as an active ingredient.

[0082] In the present invention, the terms "saikosaponin C", "food-logically acceptable salt", and "food composition" are as described above.

[0083] The symptom caused by vascular aging may be arteriosclerosis.

[0084] In addition, the symptoms due to vascular aging or symptoms due to 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 in 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 monocyte-vascular endothelial cell adhesion, increased NLRP3 inflammasome activity, increased macrophage foam cell formation, and increased macrophage foam cell formation by NLRP3 inflammasome.

[0085] A food composition containing saikosaponin C or a food-based acceptable salt thereof of the present invention as an active ingredient has the effect of improving or preventing symptoms caused by vascular aging as described above.

[0086]

[0087] In another aspect, the present invention provides a use of Saikosaponin C or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of a disease caused by vascular aging.

[0088] In another aspect, the present invention provides use of saikosaponin C or a food-based acceptable salt thereof for the production of a food for improving or preventing a disease caused by vascular aging.

[0089] In another aspect, the present invention provides a use of Saikosaponin C or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of symptoms due to vascular aging.

[0090] In another aspect, the present invention provides use of saikosaponin C or a food-based acceptable salt thereof for the manufacture of a food for improving or preventing symptoms caused by vascular aging.

[0091] In another aspect, the present invention provides a method for treating or preventing a disease caused by vascular aging or a symptom caused by vascular aging, comprising the step of administering Saikosaponin C or a pharma- ceutically acceptable salt thereof to an individual.

[0092] In the present invention, the terms "saikosaponin C," "pharmaceutical acceptable salt," "food-based acceptable salt," "disease caused by vascular aging," "symptoms caused by vascular aging," "pharmaceutical composition," and "food composition" are as described above.

[0093] In the present invention, the term "individual" refers to any animal, including humans, that develops a disease or symptoms caused by vascular aging. This term includes, without limitation, mammals and birds, including cows, pigs, sheep, chickens, dogs, humans, etc., and includes individuals whose disease or symptoms caused by vascular aging are suppressed by the Saikosaponin C or a pharma- ceutically acceptable salt thereof, and thus whose disease or symptoms caused by vascular aging are treated.

[0094] EXAMPLES

[0095] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings, in order to enable those skilled in the art to easily carry out the present invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments set forth 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 family. The molecular weight of Saikosaponin C is 927.12 and the chemical formula is C 48 H 78 O 17 The compound was used in the following experiments at concentrations of 5 μM and 20 μM and was purchased from Abcam (Cambridge, MA, USA).

[0099]

[0100] [Experimental Example]

[0101] Experimental Example 1. Effect of Saikosaponin C on alleviating the inhibition of vascular endothelial cell growth induced by high glucose concentration (substance metabolism analysis - MTT assay)

[0102] We developed a cell-level high fat diet evaluation system using 33 mM high glucose (HG), which is six times higher than the standard glucose concentration of 5.5 mM in vascular endothelial cell culture medium.

[0103] Specifically, two types of vascular endothelial cells, HUVEC (human umbilical vein endothelial cells) and HPAEC (human pulmonary artery endothelial cells), were pretreated with Saikosaponin C (SSc) at concentrations of 5μM and 20μM for 2 hours, and then treated with high-concentration glucose at 33mM for 72 hours. Cell growth was then analyzed by the MTT assay, which measures the mitochondrial substance replacement capacity of living cells.

[0104] Figure 1 shows the MTT assay to evaluate whether Saikosaponin C alleviates the growth inhibition of vascular endothelial cells (HUVEC, HPAEC) caused by high-concentration glucose, which corresponds to a high-fat diet. The growth of vascular endothelial cells was evaluated by measuring the absorbance of MTT formazan at a wavelength of 590 nm.

[0105] As a result, the growth of HUVEC and HPAEC was inhibited by high-concentration glucose treatment. However, the inhibition of cell growth caused by high-concentration glucose was alleviated in HUVEC and HPAEC pretreated with Saikosaponin C. In particular, it was confirmed that the effect of alleviating the inhibition of cell growth increased as the concentration of Saikosaponin C increased.

[0106]

[0107] Experimental Example 2. Effect of Saikosaponin C on alleviating the inhibition of vascular endothelial cell growth induced by high glucose concentration (cell count analysis - Trypan blue staining)

[0108] As in Experimental Example 1, a high-fat diet evaluation system at the cellular level was constructed.

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

[0110] Figure 2 shows the effect of Saikosaponin C on the suppression of vascular endothelial cell (HUVEC, HPAEC) growth caused by high-concentration glucose, which corresponds to a high-fat diet, evaluated by trypan blue staining. The growth of vascular endothelial cells was evaluated by observing the cell number using the trypan blue staining method.

[0111] As a result, the growth of HUVEC and HPAEC was inhibited by high-concentration glucose treatment. However, the inhibition of cell growth caused by high-concentration glucose was alleviated in HUVEC and HPAEC pretreated with Saikosaponin C. In particular, it was confirmed that the effect of alleviating the inhibition of cell growth increased as the concentration of Saikosaponin C increased.

[0112]

[0113] Experimental Example 3. Analysis of the effect of Saikosaponin C in alleviating vascular endothelial cell senescence induced by high glucose (SA-β-gal staining and vascular senescence marker protein expression analysis)

[0114] As in Experimental Example 1, a high-fat diet evaluation system at the cellular level was constructed.

[0115] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pretreated with Saikosaponin C at concentrations of 5μM and 20μM for 2 hours, and then treated with high-concentration glucose at 33mM for 72 hours (for SA-β-gal staining) or 48 hours (for vascular senescence marker protein analysis). The degree of cellular senescence was then measured by SA-β-gal staining, and the presence or absence of senescence was measured by the density of blue-stained cells (SA-β-gal positive) out of the total cells. In addition, 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 SA-β-gal (senescence-associated β-galactosidase) staining and Western blotting to evaluate whether Saikosaponin C alleviates the aging of vascular endothelial cells (HUVEC, HPAEC) caused by high glucose concentration, which corresponds to a high-fat diet. Specifically, the aging 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 (Figures 3(c) and 3(d)), and the results are shown in graph form (Figures 3(a) and 3(b)). Figures 3(e) and 3(f) show the results of Western blotting to confirm the expression of vascular aging marker proteins such as PAI-1, ICAM-1, and VCAM-1 in two types of vascular endothelial cells (HUVEC, HPAEC).

[0117] As a result, high glucose treatment increased the density of blue-stained cells (SA-β-gal positive) in HUVEC and HPAEC (Figure 3(a), (b), (c), and (d)) and the expression of vascular aging marker proteins (Figure 3(e) and (f)), confirming that high glucose induces vascular cell aging. However, in HUVEC and HPAEC pretreated with Saikosaponin C, the number of SA-β-gal positive cells and the expression of PAI-1, ICAM-1, and VCAM-1 were reduced, confirming that vascular cell aging caused by high glucose was significantly reduced. In particular, it was confirmed that the inhibitory effect on vascular cell aging increased as the concentration of Saikosaponin C increased.

[0118]

[0119] Experimental Example 4. Analysis of the effect of Saikosaponin C in alleviating the G1 growth delay induced by high glucose (cell cycle analysis and cell cycle regulatory protein expression analysis)

[0120] As in Experimental Example 1, a high-fat diet evaluation system at the cellular level was constructed.

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

[0122] Fig. 4 shows the results of cell cycle analysis and the measurement of the expression of G1 growth arrest-inducing proteins (p21, p27, p16) to evaluate whether Saikosaponin C alleviates the G1 growth arrest of vascular endothelial cells (HUVEC, HPAEC) caused by high concentration glucose, which corresponds to a high fat diet. Since cell senescence is closely related to the G1 growth arrest of the cell cycle, the cell cycle was evaluated using a flow cytometer (Fig. 4(a) and Fig. 4(b)). In addition, the expression of p21, p27, and p16, which are G1 growth arrest-inducing proteins, was measured by Western blotting (Fig. 4(c) and Fig. 4(d)).

[0123] As a result, it was confirmed that high-concentration glucose treatment delayed G1 phase growth in HUVEC and HPAEC, as well as increased expression of p21, p27, and p16 proteins. However, in HUVEC and HPAEC pretreated with Saikosaponin C, high-concentration glucose-induced G1 phase growth delay and increased expression of p21, p27, and p16 proteins were suppressed, and it was confirmed that the effect of suppressing G1 phase growth delay and the increase in expression of proteins that induce G1 phase growth delay increased with increasing concentration of Saikosaponin C. G1 phase growth delay is a phenomenon seen in the cellular aging process, and these results support the anti-vascular aging efficacy of Saikosaponin C.

[0124]

[0125] Experimental Example 5. Analysis of the effect of Saikosaponin C in mitigating the generation of reactive oxygen species induced by high glucose (fluorescence image analysis)

[0126] As in Experimental Example 1, a high-fat diet evaluation system at the cellular level was constructed.

[0127] Specifically, two types of vascular endothelial cells, HUVEC and HPAEC, were pretreated with Saikosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with high-concentration glucose at 33 mM for 24 hours. After that, hydrogen peroxide (H2O2) and superoxide anion (O2) generated from the cytoplasm and mitochondria were ·- ) was measured using each fluorescent probe. In addition, a sample treated with 100 μM hydrogen peroxide for 1 hour, then treated for another 23 hours after medium change was used as a positive control for reactive oxygen species induction.

[0128] Different analytical reagents were used depending on the type of reactive oxygen species and the intracellular location where they were generated. Specifically, H2DCFDA (2,7-dichlorodihydrofluorescein diacetate), DHE (dihydroethidium), DHR (dihydrorhodamine 123), and Mito-SOX reagents were used to analyze intracytoplasmic hydrogen peroxide, intracytoplasmic superoxide anion, mitochondrial hydrogen peroxide, and mitochondrial superoxide anion, respectively. The 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 fluorescence image analysis to evaluate whether Saikosaponin C reduces the generation of reactive oxygen species (ROS) in vascular endothelial cells (HUVEC, HPAEC) caused by high glucose concentration in a high fat diet. Figure 5B shows the results of the fluorescence image analysis, and Figure 5A shows the results in graph form.

[0130] As a result, it was confirmed that high-concentration glucose treatment increased the production of various reactive oxygen species such as intracytoplasmic hydrogen peroxide, intracytoplasmic superoxide anion, mitochondrial hydrogen peroxide, and mitochondrial superoxide anion in HUVEC and HPAEC. However, in HUVEC and HPAEC pretreated with Saikosaponin C, the production of various reactive oxygen species caused by high-concentration glucose was reduced, and the inhibitory effect on the production of reactive oxygen species increased as the concentration of Saikosaponin C increased. Since hydrogen peroxide and superoxide anion are representative reactive oxygen species, these results support the anti-vascular aging efficacy of Saikosaponin C based on ROS scavenging.

[0131]

[0132] Experimental Example 6. Analysis of the effect of Saikosaponin C in alleviating the decrease in NOS-NO signaling induced by high glucose (analysis of nitric oxide concentration, nitric oxide synthase activity and protein expression)

[0133] As in Experimental Example 1, a high-fat diet evaluation system at the cellular level was constructed.

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

[0135] Figure 6 shows the evaluation of whether Saikosaponin C alleviates the decrease in NOS-NO signaling in vascular endothelial cells (HUVEC, HPAEC) caused by high-concentration glucose, which corresponds to a high-fat diet. Nitric oxide generation was measured using a Nitric Oxide Assay Kit (Figure 6(a) and Figure 6(b)). Specifically, nitrate (NO3 -) and nitrite (nitrite, NO2 - The values ​​of nitrate reductase and Griess reagent were analyzed. Nitric oxide synthase activity was measured using a NOS Activity Assay Kit (Figure 6(c) and (d)), and the expression and phosphorylation (Ser114) of nitric oxide synthase were measured by Western blotting (Figure 6(e) and (f)).

[0136] As a result, it was confirmed that high glucose treatment reduced nitric oxide (NO) levels as well as the enzyme activity, protein expression and phosphorylation of Ser114 residue (p-eNOS) of nitric oxide synthase in HUVEC and HPAEC. However, the reduction in NOS-NO signaling caused by high glucose was alleviated in HUVEC and HPAEC pretreated with Saikosaponin C, and the effect of alleviating the reduction in NOS-NO signaling increased as the concentration of Saikosaponin C increased. Nitric oxide generated by nitric oxide synthase is a vasodilator that induces vascular relaxation, unlike reactive oxygen species, and helps maintain healthy blood vessels, so these results support the anti-vascular aging efficacy of Saikosaponin C.

[0137]

[0138] Experimental Example 7. Analysis of the effect of Saikosaponin C in alleviating 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 high-fat diet evaluation system at the cellular level was constructed.

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

[0141] Figure 7 shows whether Saikosaponin C alleviates Sirt1 activity, protein expression and phosphorylation in vascular endothelial cells (HUVEC, HPAEC) fed a high-fat diet with high glucose. Sirt1 activity was measured using a Sirt1 activity assay kit (Figure 7(a) and (b)), and Sirt1 protein expression and phosphorylation (Ser27, Ser47) were measured using Western blotting (Figure 7(c) and (d)).

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

[0143]

[0144] Experimental Example 8. Analysis of the mechanism of action of Saikosaponin C in alleviating endothelial cell senescence induced by high glucose (SA-β-gal staining, nitric oxide synthase activity and Sirt1 enzyme activity analysis)

[0145] As in Experimental Example 1, a high-fat diet evaluation system at the cellular level was constructed.

[0146] Specifically, vascular endothelial cells (HUVECs) were pretreated with Saikosaponin 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). SA-β-gal staining, nitric oxide synthase activity, and Sirt1 enzyme activity were then analyzed. To analyze whether nitric oxide synthase and Sirt1 protein are involved in the anti-vascular aging efficacy of Saikosaponin C as well as the connection between nitric oxide synthase and Sirt1 protein, L-NAME (LNAME), an inhibitor of nitric oxide synthase, was used. G -Nitro arginine methyl ester (5 mM) and EX-527 (10 μM), an inhibitor of Sirt1 protein, were used.

[0147] Figure 8 shows whether nitric oxide synthase and Sirt1 protein are involved in the efficacy of Saikosaponin C in alleviating aging of vascular endothelial cells (HUVEC) caused by high glucose concentration, which corresponds to a high-fat diet, and evaluates the relationship between nitric oxide synthase and Sirt1 protein. Vascular endothelial cell aging was evaluated by intracellular SA-β-gal staining (Figure 8(a)), and nitric oxide synthase (Figure 8(b)) and Sirt1 protein activities (Figure 8(c)) were measured using a NOS activity assay kit and a Sirt1 activity assay kit, respectively.

[0148] As a result, as seen in Figure 3, high-concentration glucose treatment increased HUVEC senescence (SA-β-gal positive cells), and pretreatment with Saikosaponin C alleviated HUVEC senescence. However, it was confirmed that the anti-vascular aging effect of Saikosaponin C was significantly inhibited in HUVECs pretreated with L-NAME, an inhibitor of nitric oxide synthase, or EX-527, an inhibitor of Sirt1 protein. It was also confirmed that both L-NAME and EX-527 pretreated HUVECs inhibited the alleviation effect of Saikosaponin C on the decrease in nitric oxide synthase (NOS) activity, but L-NAME pretreatment could not inhibit the alleviation effect of the decrease in Sirt1 protein activity caused by Saikosaponin C.

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

[0150]

[0151] Experimental Example 9. Inhibitory effect of Saikosaponin C on monocyte-endothelial cell adhesion induced by TNF-α during atherosclerosis (fluorescence analysis and protein expression analysis)

[0152] From the above Experimental Example 3, it was confirmed that Saikosaponin C inhibits vascular endothelial cell aging. Since arteriosclerosis is a representative disease caused by vascular aging, we next attempted to clarify whether Saikosaponin C exhibits arteriosclerosis inhibitory ability. The first arteriosclerosis analysis was performed by evaluating monocyte-vascular endothelial cell adhesion, which plays an important role in the early stage of arteriosclerosis pathogenesis.

[0153] Specifically, two types of vascular endothelial cells, HUVEC (human umbilical vein endothelial cells) and HCAEC (human coronary artery endothelial cells), were pretreated with Saikosaponin C at concentrations of 5 μM and 20 μM for 2 hours, and then treated with TNF-α (20 ng / ml) for 8 hours. Then, monocyte-vascular endothelial cell adhesion and vascular endothelial cell adhesion molecule (ICAM-1, VCAM-1) expression were observed. The human mononuclear cell line, THP-1, was used as the mononuclear cell line, and the THP-1 cells were inoculated with the green fluorescent substance Calcein AM (1×10 6After leveling at 1 μg / ml per cell for 30 minutes, the cells were spread on the two types of vascular endothelial cells. Monocyte-vascular endothelial cell adhesion was evaluated by imaging analysis using a confocal microscope and fluorescence analysis using a microplate reader, and adhesion protein (ICAM-1, VCAM-1) expression was evaluated by Western blotting.

[0154] Figure 9 shows whether Saikosaponin C inhibits monocyte-endothelial cell adhesion caused by TNF-α, an arteriosclerosis inducer. Calcein AM fluorescence analysis was performed using a confocal microscope and a microplate reader, and the expression of cell adhesion proteins (ICAM-1, VCAM-1) was evaluated by Western blotting.

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

[0156] As a result, it was confirmed that TNF-α treatment increased monocyte-endothelial cell adhesion in both types of vascular endothelial cells (HUVEC, HCAEC). However, it was confirmed that such adhesion was inhibited when the cells were pretreated with Saikosaponin C. It was also confirmed that Saikosaponin C inhibited the expression of adhesion proteins in vascular endothelial cells involved in monocyte-endothelial cell adhesion. Monocyte-endothelial cell adhesion is considered to be an early stage in the development of arteriosclerosis, and adhesion proteins such as ICAM-1 and VCAM-1 are also vascular aging marker proteins, so these results indicate that Saikosaponin C has anti-vascular aging efficacy and therefore anti-atherosclerosis efficacy.

[0157]

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

[0159] As in Experiment 9, we attempted to determine whether Saikosaponin C exhibits arteriosclerosis-suppressing ability. The second arteriosclerosis assay was performed by evaluating the non-ideal proliferation and migration of vascular smooth muscle cells, which are the main mechanisms signaling 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 Saikosaponin C at concentrations of 5μM and 20μM for 2 hours, and then treated with PDGF-BB (20ng / ml), a platelet-derived growth factor (PDGF), for 24 hours. The proliferation and migration of vascular smooth muscle cells were then 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 migration ability of vascular smooth muscle cells.

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

[0162] As a result, it was confirmed that PDGF-BB treatment increased the proliferation and migration of vascular smooth muscle cells in both types of vascular smooth muscle cells (AoSMC, CASMC). Unlike previous Experimental Example 9, it was confirmed that pretreatment with Saikosaponin C did not inhibit such proliferation and migration. These results indicate that the anti-atherosclerotic effect of Saikosaponin C is not related to the proliferation and migration of vascular smooth muscle cells.

[0163]

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

[0165] As shown in Experimental Examples 9 and 10, Saikosaponin C inhibited monocyte-endothelial cell adhesion induced by TNF-α, but did not inhibit vascular smooth muscle cell abnormal proliferation and migration induced by PDGF. Therefore, we attempted to clarify whether it exhibits atherosclerosis suppression ability using another atherosclerosis phenotype analysis. The third atherosclerosis analysis was performed by observing macrophage foam cell formation, which plays an important role in the early stage of atherosclerosis as well as monocyte-endothelial cell adhesion. Macrophage foam cell formation is a step between monocyte-endothelial cell adhesion and non-ideal proliferation and migration of vascular smooth muscle cells in the progression to atherosclerosis.

[0166] Specifically, THP-1 cells, a mononuclear cell line, were differentiated into macrophages by treating them with PMA (Phorbol 12-myristate 13-acetate) at a concentration of 150 nM for 48 hours. The differentiated macrophages were pretreated with Saikosaponin 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. The formation of foam cells from macrophages was then observed. Foam cell formation was evaluated by microscopic analysis after staining with Oil Red O, which adsorbs to lipid molecules, and absorbance analysis using a microplate reader.

[0167] FIG. 11 shows whether Saikosaponin C inhibits macrophage foam cell formation caused by oxidized LDL (ox-LDL), an arteriosclerosis inducer. FIG. 11 shows the results of analyzing the degree of macrophage foam cell formation after cells fixed with 10% formalin for 30 minutes were stained with Oil Red O for 30 minutes. Nuclei were stained with hematoxylin for 1 minute. The degree of foam cell formation was analyzed by observing the degree of red coloring by Oil Red O stain under a microscope as shown in FIG. 11(a) or by measuring absorbance at 540 nm using a microplate reader after eluting Oil Red O stain with 100% isopropanol for 10 minutes as shown in FIG. 11(b).

[0168] As a result, it was confirmed that macrophages take in large amounts of lipids and transform into foam cells when treated with oxidized LDL. However, it was confirmed that pretreatment with Saikosaponin C inhibited the formation of foam cells caused by lipid deposition. Since the transformation of macrophages into foam cells, along with monocyte-endothelial cell adhesion, is considered a typical pathological finding of early arteriosclerosis, these results indicate that Saikosaponin C acts at the early stage of arteriosclerosis and has anti-atherosclerotic effects.

[0169]

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

[0171] As in Experimental Examples 9, 10, and 11, we tried to clarify whether Saikosaponin C has the ability to inhibit arteriosclerosis. NLRP3 (NOD-like receptor protein-3), which plays the role of a sensor, forms a protein complex called NLRP3 inflammasome together with ASC, which plays the role of an adaptor, and caspase-1, which plays the role of an effector. NLRP3 inflammasome causes pyroptosis, which is a cell death associated with inflammation, by secreting IL-1β, an inflammatory cytokine, and activating caspase-1 in response to infection and cell damage. Above all, NLRP3 inflammasome is known to promote macrophage foam cell formation by secreting IL-1β and activating caspase-1. Since Saikosaponin C showed the effect of inhibiting macrophage foam cell formation caused by oxidized LDL in Experimental Example 11, the fourth arteriosclerosis analysis was performed by evaluating the activity of NLRP3 inflammasome, which promotes macrophage 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 and differentiated into macrophages. The differentiated macrophages were pretreated with saikosaponin at a concentration of 20 μM for 2 hours and then treated with monosodium urate crystals (MSU), an NLRP3 inflammasome inducer (activator), at a concentration of 200 μg / ml for 6 hours. IL-1β secretion and caspase-1 cleavage were then analyzed. In addition, as a control group to compare the efficacy of saikosaponin C, the NLRP3 inflammasome inhibitor MCC950 (1 μM) was pretreated for 2 hours in the same manner as saikosaponin C.

[0173] Figure 12 evaluates whether Saikosaponin C inhibits NLRP3 inflammasome activity, which promotes macrophage foam cell formation seen in early atherosclerosis. 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 by Western blotting.

[0174] As a result, we confirmed that uric acid crystal (MSU) treatment increased IL-1β secretion and caspase-1 cleavage. However, when Saikosaponin C was pretreated, as with MCC950, an NLRP3 inflammasome inhibitor, it was confirmed that such IL-1β secretion and caspase-1 cleavage were suppressed. Caspase-1 cleavage and subsequent IL-1β secretion are required for NLRP3 inflammasome activation, and the effect of suppressing IL-1β secretion and caspase-1 cleavage means that Saikosaponin C can suppress NLRP3 inflammasome activity. In addition, since activated NLRP3 inflammasome promotes foam cell formation, these results further prove the anti-atherosclerotic efficacy of Saikosaponin C in the early stage of atherosclerosis by inhibiting macrophage foam cell formation.

[0175]

[0176] In summary, Saikosaponin C inhibited vascular aging caused by vascular endothelial cell senescence, and inhibited monocyte-vascular endothelial cell adhesion and macrophage foam cell formation by NLRP3 inflammasome, which play important roles in the early stage of arteriosclerosis pathogenesis. Therefore, Saikosaponin C can be used as an arteriosclerosis treatment based on the inhibition of vascular aging. Such a new concept arteriosclerosis treatment based on the inhibition of vascular aging can be used alone for arteriosclerosis disease to show an independent therapeutic effect, or can be used in combination with existing arteriosclerosis treatments that lower LDL-C to enhance the therapeutic effect.

[0177]

[0178] Although the 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 made by those skilled in the art using the basic concept of the present invention defined in the appended claims also fall within the scope of the present invention.

Claims

1. A pharmaceutical composition for treating or preventing diseases caused by vascular aging, comprising saikosaponin C or a pharma- ceutical acceptable salt thereof as an active ingredient.

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

3. The pharmaceutical composition for treating or preventing a disease caused by vascular aging according to claim 1 , wherein the vascular aging is aging of vascular endothelial cells.

4. The pharmaceutical composition for treating or preventing a disease caused by vascular aging according to claim 1, wherein the composition inhibits monocyte-endothelial cell adhesion, NLRP3 inflammasome activity, macrophage foam cell formation, or macrophage foam cell formation caused by NLRP3 inflammasome.

5. A food composition for improving or preventing diseases caused by vascular aging, comprising saikosaponin C or a nutrient-acceptable salt thereof as an active ingredient.

6. The food composition for improving or preventing diseases caused by vascular aging according to claim 5 , wherein the disease caused by vascular aging is arteriosclerosis.

7. The food composition for improving or preventing diseases caused by vascular aging according to claim 5 , wherein the vascular aging is aging of vascular endothelial cells.

8. The food composition for improving or preventing a disease caused by vascular aging according to claim 5, wherein the composition inhibits monocyte-endothelial cell adhesion, NLRP3 inflammasome activity, macrophage foam cell formation, or macrophage foam cell formation by NLRP3 inflammasome.

9. The food composition for improving or preventing diseases caused by vascular aging according to claim 5, wherein the food composition is a functional health food.

10. A pharmaceutical composition for treating or preventing symptoms caused by vascular aging, comprising saikosaponin C or a pharma- ceutical acceptable salt thereof as an active ingredient.

11. The pharmaceutical composition for treating or preventing symptoms caused by vascular aging according to claim 10, wherein the symptoms caused by vascular aging are arteriosclerosis.

12. The pharmaceutical composition for treating or preventing symptoms caused by vascular aging according to claim 10, wherein the symptoms caused by vascular aging are one or more selected from the group consisting of inhibition of vascular endothelial cell growth, increased vascular endothelial cell senescence, increased production of reactive oxygen species in 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 monocytes and vascular endothelial cells, increased activity of NLRP3 inflammasome, increased formation of macrophage foam cells, and increased formation of macrophage foam cells by NLRP3 inflammasome.

13. A food composition for improving or preventing symptoms caused by vascular aging, comprising saikosaponin C or a nutrient-acceptable salt thereof as an active ingredient.

14. The food composition for improving or preventing a symptom caused by vascular aging according to claim 13, wherein the symptom caused by vascular aging is arteriosclerosis.

15. The food composition for improving or preventing symptoms caused by vascular aging according to claim 13, wherein the symptoms caused by 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 in 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 monocytes and vascular endothelial cells, increased activity of NLRP3 inflammasome, increased formation of macrophage foam cells, and increased formation of macrophage foam cells by NLRP3 inflammasome.

16. The food composition for improving or preventing symptoms caused by vascular aging according to claim 13, wherein the food composition is a functional health food.

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