Composition containing gintonin for the prevention and treatment of ischemic cerebrovascular disease

Abstract

The present invention relates to a composition containing gintonin as an active ingredient for the prevention and treatment of ischemic cerebrovascular disorders, including cerebral infarction. The gintonin of the present invention can reduce the volume of cerebral infarction and increase survival rates after cerebral infarction, making it useful for the prevention and treatment of cerebral infarction. Furthermore, pretreatment of human brain microvascular endothelial cells (HBMECs) with gintonin has been shown to have neuroprotective and injury-preventing effects, while posttreatment with gintonin has been shown to have neuroprotective and therapeutic effects. Mechanism verification revealed that gintonin is effective in restoring reduced nitric oxide (NO) levels, which are known to be involved in the onset and pathophysiology of cerebral infarction. Therefore, the use of the present invention may improve the prevention and treatment of ischemic cerebrovascular disorders, including cerebral infarction, and reduce medical costs.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2022-0186469, filed on December 27, 2022, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a composition containing gintonin as an active ingredient for the prevention and treatment of ischemic cerebrovascular disorders, including cerebral infarction. Gintonin according to the present invention has the effect of preventing cerebral infarction, reducing the volume of cerebral infarction, and increasing the survival rate after cerebral infarction, and is therefore useful for the prevention and treatment of ischemic cerebrovascular disorders.

[0003] This invention was completed with support from the Korea Research Foundation under project number 2021R1A6A1A03038865 (project specific number 1345347342). [Background technology]

[0004] Cerebral infarction is an ischemic disease in which blood vessels in the brain become blocked, resulting in necrosis of brain tissue. It is the most common type of stroke and, along with cerebral hemorrhage, is a major cause of cerebrovascular disease and is known as one of the leading causes of death worldwide.

[0005] According to the Global Burden of Disease report, stroke was the third leading cause of death and disability in the world in 2019, and is a serious disease that costs approximately $891 billion (approximately 1,100 trillion won).

[0006] According to the results of a study of 11,136 acute stroke patients admitted to 14 general hospitals across Korea from 2011 to 2013, the average medical expenses paid per year before the stroke was approximately 7.6 million won, but in the first year after the stroke, this amount increased by more than fourfold to approximately 33 million won.The study also found that the total medical expenses paid by a stroke patient over five years averaged approximately 100 million won.

[0007] Additionally, it was found that patients who fully recovered without any aftereffects three months after discharge paid a total medical expense of approximately 47 million won over five years, while patients who were unable to walk or perform daily activities without assistance paid a total of 240 million won, approximately five times as much. This suggests that the way in which stroke patients are treated in the acute phase can significantly affect not only the degree of recovery but also the economic burden. Therefore, from the perspective of reducing national medical expenses, the prevention and treatment of stroke is extremely important.

[0008] With the aging of the population and changes in dietary habits, the incidence of stroke is increasing year by year, further increasing the global burden. Primary and secondary brain damage caused by stroke result in complex pathophysiological processes, including inflammation, neuronal cell death, ischemia-reperfusion injury, blood-brain barrier damage, neurotoxic substance release, vitreous formation, oxidative stress, and cerebral edema. Currently, treatments for ischemic stroke primarily include thrombectomy, stenting and angioplasty, surgical treatment (compressive craniectomy and carotid artery distal resection), thrombolytic agents, and rehabilitation training. These treatments can, to some extent, improve the prognosis and quality of life of stroke patients. While thrombolysis and mechanical thrombectomy have significantly improved stroke prognosis, the majority of patients remain unable to lead independent lives, even with these treatments. Therefore, the development of new stroke treatments is essential.

[0009] As mentioned above, the current standard treatment for acute cerebral infarction involves administering a thrombolytic agent within 4.5 hours of onset or performing thrombectomy within 24 hours of onset to reopen blocked cerebral blood vessels. However, even after reopening, many patients die or become disabled due to brain cell death caused by reperfusion injury, inflammation, and delayed death. Therefore, research is underway to develop new treatments, including methods to enhance the efficacy of thrombolytic agents, reconstruct blood vessels, and reduce inflammatory (secondary) brain damage.

[0010] Gintonin is a novel glycolipoprotein extracted from ginseng. It is a non-ginsenoside substance distinct from well-known saponins. Its main components are lysophosphatidic acid (LPA) and other proteins. The LPA receptor (LPAR) is the specific target receptor for gintonin, with high affinity. Gintonin is therefore an LPAR ligand derived from ginseng. Previous studies have demonstrated the beneficial role of gintonin in mouse models of chronic degenerative neurological diseases such as Alzheimer's disease and Parkinson's disease. Active research into the efficacy of gintonin is also underway. However, research and validation into the treatment of cerebral infarction has yet to be conducted.

[0011] Nitric oxide (NO) is a gas naturally produced in the human body and a crucial signaling molecule that plays a wide variety of roles in bodily functions. One of its most important functions is vasodilatory function, relaxing and dilating blood vessels, increasing blood flow and thus playing a key role in maintaining healthy blood pressure and circulation. Nitric oxide also regulates the immune system, performing immune functions to prevent infection, and is involved in various cell signaling pathways that regulate various cell signaling functions such as cell growth, differentiation, and cell death. The main physiological functions of nitric oxide include maintaining vascular tone, reducing inflammatory responses, maintaining thrombolytic homeostasis, and regulating cell growth.

[0012] Nitric oxide also plays an important role in brain development and function, regulating blood flow to the brain, protecting nerve cells from damage, and promoting learning and memory. Nitric oxide also helps maintain cardiovascular health by preventing blood clots and keeping blood vessels healthy, and is used to treat cardiovascular diseases such as erectile dysfunction.

[0013] There are three types of nitric oxide synthase (NOS): eNOS, the most common type of NOS, is found in endothelial cells lining blood vessels and is responsible for producing nitric oxide, which causes vasodilation; iNOS is found in immune cells and is involved in inflammatory responses; and nNOS is found in neurons and is involved in learning and memory.

[0014] Nitric oxide (NO) is a very important molecule, but when overproduced it can be harmful. For example, high levels of NO can damage cells and tissues. This can occur in situations such as sepsis and inflammation.

[0015] Overall, nitric oxide is a versatile molecule that plays a vital role in various bodily functions, so maintaining a proper balance is important. As mentioned above, nitric oxide is a substance that plays an important role in dilating blood vessels and improving blood circulation. When cerebral infarction occurs, the production of nitric oxide in brain tissue decreases. This can reduce cerebral blood flow and exacerbate brain damage.

[0016] Korean Patent Registration No. 10-2011-0128734 discloses "gintonin that inhibits cancer metastasis and a composition for anti-cancer and cancer metastasis inhibition containing said gintonin as an active ingredient," and Korean Patent Registration No. 10-1077226-0000 discloses "a composition for preventing and treating degenerative nervous system diseases containing gintonin as an active ingredient." However, there has never been any disclosure of a composition containing gintonin as an active ingredient for preventing and treating cerebral infarction or ischemic cerebral infarction, as in the present invention. Summary of the Invention [Problem to be solved by the invention]

[0017] As mentioned above, the present invention is highly effective in preventing and treating ischemic cerebrovascular disorders, including cerebral infarction. After researching substances that are easily accessible to patients, the present inventors confirmed the therapeutic effect of gintonin on a mouse model of focal ischemic stroke, leading to the completion of the present invention.

[0018] Therefore, an object of the present invention is to provide a composition containing gintonin as an active ingredient, which is highly effective in preventing and treating ischemic cerebrovascular disorders including cerebral infarction. [Means for solving the problem]

[0019] The present invention provides a pharmaceutical composition containing gintonin for preventing or treating ischemic cerebrovascular disease.

[0020] According to a preferred embodiment of the present invention, the gintonin is lysophosphatidic acids in gintonin.

[0021] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder is ischemic cerebral infarction.

[0022] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder is caused by a decrease in production of nitric oxide.

[0023] According to a preferred embodiment of the present invention, the composition reduces the volume of cerebral infarction.

[0024] According to a preferred embodiment of the present invention, the composition increases the survival rate of an individual who has suffered from cerebral infarction.

[0025] The present invention also provides a health functional food composition containing gintonin for preventing or improving ischemic cerebrovascular disease.

[0026] The present invention also provides a composition for protecting nerve cells, which comprises gintonin.

[0027] According to a preferred embodiment of the present invention, the gintonin is lysophosphatidic acids in gintonin.

[0028] According to a preferred embodiment of the present invention, the nerve cells are nerve cells damaged by reduced production of nitric oxide.

[0029] According to a preferred embodiment of the present invention, the nerve cells are cerebrovascular nerve cells.

[0030] The present invention also provides a functional health food composition for protecting nerve cells, which contains gintonin.

[0031] The present invention also provides a method for treating ischemic brain disease, comprising administering a composition containing gintonin to a patient suffering from ischemic cerebrovascular disease.

[0032] The present invention also provides a use of gintonin for the treatment of ischemic cerebrovascular disease.

[0033] The present invention also provides a use of gintonin for the manufacture of a therapeutic agent for ischemic cerebrovascular disease.

[0034] The term "prevention" in the present invention means any action to suppress ischemic cerebrovascular disorders or related diseases or to delay their onset by the gintonin of the present invention.

[0035] "Improvement" or "treatment" in the present invention means any action that improves or benefits the parameters associated with ischemic cerebrovascular disease or related diseases, such as the severity of symptoms, using gintonin of the present invention.

[0036] The present invention relates to the preventive, ameliorative or therapeutic effects of gintonin on ischemic cerebrovascular disorders including cerebral infarction, and to the neuroprotective effects of gintonin. The gintonin may refer to gintonin isolated from white ginseng, red ginseng or red ginseng pomace.

[0037] We investigated the neuroprotective effects of gintonin in a mouse model of focal ischemic stroke. First, C57BL / 6N mice were orally administered saline (n = 50), gintonin at 150 mg / kg (n = 65), or 300 mg / kg (n = 20) once daily for 1 week. Furthermore, 15 randomly selected mice from the 150 mg / kg group were intraperitoneally injected with an LPAR antagonist (Ki16425, 30 mg / kg) before oral administration of gintonin. Cerebral autoregulatory dysfunction (CAD) was induced in all mice by intraperitoneal administration of an NOS antagonist, followed by hemispheric ischemia by common carotid artery occlusion (CCAO). Survival rates and final infarct volumes after 1 week were compared between groups (log-rank test and Student's t-test, respectively). Gintonin 150 mg / kg (vs. saline) increased the 1-week survival rate (35 / 50 [70%] vs. 23 / 50 [46%], p=0.009) and reduced infarct size (92.20 ± 14.53 mm 3 vs. 138.70±16mm 3 , p = 0.005). Gintonin 300 mg / kg tended to increase survival rate (12 / 20 [60%], p = 0.18 vs. saline) to a non-significant level, but did not significantly decrease or increase infarct size (40 ± 36 mm3, p = 0.75 vs. saline) (Figure 1). Pretreatment with an LPA receptor antagonist inhibited the reduction in post-stroke survival rate (6 / 15 [40%], p = 0.61 vs. saline) and infarct size (183.81 ± 31.97 mm3, p = 0.88 vs. saline) induced by gintonin 150 mg / kg (Figure 2). In conclusion, gintonin 150 mg / kg increased post-stroke survival rate and reduced infarct volume, likely due to LPAR inhibition.

[0038] Based on these in-vivo animal model results, we conducted cell experiments using HBMEC (Human Brain Microvascular Endothelial Cells) to verify the mechanism of nitric oxide involvement in the preventive, ameliorative, or therapeutic effects of gintonin on cerebral infarction. We confirmed that gintonin restored nitric oxide levels that had been reduced by treatment with the nitric oxide synthase (NOS) inhibitor L-NAME (Figures 3-5). Furthermore, the fact that pretreatment with the LPAR antagonist Ki16425 (5 μM) did not restore nitric oxide levels reduced by gintonin (10 μg / ml) was consistent with the results of the in-vivo animal model.

[0039] Therefore, the present invention can provide a pharmaceutical composition containing gintonin for preventing or treating ischemic cerebrovascular disease.

[0040] According to a preferred embodiment of the present invention, the gintonin may be lysophosphatidic acids in gintonin.

[0041] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be ischemic cerebral infarction.

[0042] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be caused by a decrease in production of nitric oxide.

[0043] According to a preferred embodiment of the present invention, the composition may reduce the volume of cerebral infarction.

[0044] According to a preferred embodiment of the present invention, the composition may increase the survival rate of an individual who has suffered from cerebral infarction.

[0045] The pharmaceutical compositions of the present invention can be in various oral or parenteral dosage forms, and can be formulated using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants or surfactants, diluents, or excipients.

[0046] Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing one or more compounds with at least one or more excipients, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or gelatin, etc. For example, tablets or sugar-coated tablets can be obtained by blending an active ingredient with a solid excipient, pulverizing the mixture, adding appropriate excipients, and then processing the mixture into a granule mixture. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to the frequently used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives may be contained. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, sodium alginate, and the like may be added as disintegrants, and anti-coagulants, flavors, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, packaging agents, pigments, and preservatives may also be contained.

[0047] Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. 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, laurin butter, glycerol, gelatin, and the like.

[0048] The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be formulated into an injection form for intraperitoneal, intravenous, intramuscular, subcutaneous or intracerebral injection by methods known in the art.

[0049] Injectable solutions must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Suitable carriers for injectable solutions include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferred carriers include Hank's solution, Ringer's solution, triethanolamine-containing phosphate buffered saline (PBS), sterile water for injection, and isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injectable solution from microbial contamination, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal, may also be included. In addition, in many cases, the injectable solution may further contain an isotonic agent, such as sugar or sodium chloride.

[0050] The compositions of the present invention are administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the patient's disease, 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. The compositions of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and in single or multiple doses. That is, the total effective amount of the compositions of the present invention can be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy at the minimum dose without side effects, which can be easily determined by one of ordinary skill in the art.

[0051] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

[0052] The compositions of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy and other treatments using biological response modifiers.

[0053] The present invention also provides a health functional food composition containing gintonin for preventing or improving ischemic cerebrovascular disease.

[0054] According to a preferred embodiment of the present invention, the gintonin may be lysophosphatidic acids in gintonin.

[0055] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be ischemic cerebral infarction.

[0056] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be caused by a decrease in production of nitric oxide.

[0057] According to a preferred embodiment of the present invention, the composition may reduce the volume of cerebral infarction.

[0058] According to a preferred embodiment of the present invention, the composition may increase the survival rate of an individual who has suffered from cerebral infarction.

[0059] The food compositions of the present invention can be prepared in various forms by conventional methods known in the art. Examples of common foods that can be prepared by adding the gintonin of the present invention include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods (e.g., canned and bottled fruits, jams, marmalades, etc.), fish, meat and processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candy, dairy products (e.g., butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., miso paste, soy sauce, sauces, etc.). Nutritional supplements can also be prepared by adding the gintonin of the present invention to capsules, tablets, pills, etc. In addition, health functional foods include, but are not limited to, the gintonin of the present invention itself can be prepared in the form of tea, juice, and drink, and consumed in the form of liquid, granules, capsules, or powder. To use the gintonin of the present invention as a food additive, it can be prepared in the form of powder or concentrate. Furthermore, the gintonin of the present invention can be mixed with known active ingredients known to have the effect of preventing or improving ischemic cerebrovascular disease to prepare a composition.

[0060] When the gintonin of the present invention is used as a health drink, the health drink composition may contain various flavorings or natural carbohydrates as additional ingredients, as in conventional drinks. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

[0061] Furthermore, gintonin of the present invention may be contained as an active ingredient in a health functional food composition for preventing or improving ischemic cerebrovascular disorders, and the amount thereof is an amount effective for achieving the effect of preventing or improving ischemic cerebrovascular disorders, and is not particularly limited, but is preferably 0.01 to 100 wt% based on the total weight of the entire composition. The health functional food composition of the present invention can be produced by mixing gintonin with other active ingredients known to be effective against ischemic cerebrovascular disorders. In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectinic acid, pectinic acid salts, alginic acid, alginic acid salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. The health food of the present invention may also contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination.

[0062] The present invention also provides a composition for protecting nerve cells, which contains gintonin.

[0063] According to a preferred embodiment of the present invention, the gintonin may be lysophosphatidic acids in gintonin.

[0064] According to a preferred embodiment of the present invention, the nerve cells may be nerve cells damaged by decreased production of nitric oxide.

[0065] According to a preferred embodiment of the present invention, the nerve cells may be cerebrovascular-derived nerve cells.

[0066] The present invention also provides a functional health food composition for protecting nerve cells, which contains gintonin.

[0067] According to a preferred embodiment of the present invention, the gintonin may be lysophosphatidic acids in gintonin.

[0068] According to a preferred embodiment of the present invention, the nerve cells may be nerve cells damaged by decreased production of nitric oxide.

[0069] According to a preferred embodiment of the present invention, the nerve cells may be cerebrovascular-derived nerve cells.

[0070] The food composition of the present invention can be prepared in various forms by conventional methods known in the art. Examples of common foods that can be prepared by adding the gintonin of the present invention include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods (e.g., canned and bottled fruits, jams, marmalades, etc.), fish, meat and processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candy, dairy products (e.g., butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., miso, soy sauce, sauces, etc.). Nutritional supplements can also be prepared by adding the gintonin of the present invention to capsules, tablets, pills, etc. In addition, health functional foods include, but are not limited to, the gintonin of the present invention itself can be prepared in the form of tea, juice, and drink, and consumed in the form of liquid, granules, capsules, or powder. To use the gintonin of the present invention as a food additive, it can be prepared in the form of powder or concentrated liquid. Furthermore, the gintonin of the present invention can be mixed with known active ingredients known to have neuroprotective effects to prepare a composition.

[0071] When the gintonin of the present invention is used as a health drink, the health drink composition may contain various flavorings or natural carbohydrates as additional ingredients, as in conventional drinks. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention. Furthermore, gintonin of the present invention can be contained as an active ingredient in a functional health food composition for protecting nerve cells, and the amount thereof is an amount effective for achieving the neuroprotective effect and is not particularly limited, but is preferably 0.01 to 100 wt% based on the total weight of the entire composition. The functional health food composition of the present invention can be prepared by mixing gintonin with other active ingredients known to be effective in protecting nerve cells.

[0072] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectinic acid, pectinic acid salts, alginic acid, alginic acid salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. The health food of the present invention may also contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination.

[0073] The present invention also provides a method for treating ischemic brain disease, comprising administering a composition containing gintonin to a patient suffering from ischemic cerebrovascular disease.

[0074] According to a preferred embodiment of the present invention, the gintonin may be lysophosphatidic acids in gintonin.

[0075] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be ischemic cerebral infarction.

[0076] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be caused by a decrease in production of nitric oxide.

[0077] According to a preferred embodiment of the present invention, the treatment method may reduce the volume of cerebral infarction.

[0078] According to a preferred embodiment of the present invention, the treatment method may increase the survival rate of an individual who has developed cerebral infarction.

[0079] The present invention also provides a use of gintonin for treating ischemic cerebrovascular disease.

[0080] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be ischemic cerebral infarction.

[0081] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be caused by a decrease in production of nitric oxide.

[0082] According to a preferred embodiment of the present invention, the treatment may reduce the volume of cerebral infarction.

[0083] According to a preferred embodiment of the present invention, the treatment may increase the survival rate of an individual who has suffered from cerebral infarction.

[0084] According to a preferred embodiment of the present invention, the gintonin may be lysophosphatidic acids in gintonin.

[0085] The present invention also provides a use of gintonin for the manufacture of a therapeutic agent for ischemic cerebrovascular disease.

[0086] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be ischemic cerebral infarction.

[0087] According to a preferred embodiment of the present invention, the ischemic cerebrovascular disorder may be caused by a decrease in production of nitric oxide.

[0088] According to a preferred embodiment of the present invention, the therapeutic agent may reduce the volume of cerebral infarction.

[0089] According to a preferred embodiment of the present invention, the therapeutic agent may increase the survival rate of an individual who has developed cerebral infarction.

[0090] According to a preferred embodiment of the present invention, the gintonin may be lysophosphatidic acids in gintonin. [Effects of the Invention]

[0091] Gintonin, a compound containing the LPAR ligand lysophosphatidic acid receptor (LPAR) derived from ginseng, increased survival rate after stroke and reduced infarct volume in a mouse model of focal ischemic stroke. Furthermore, in a cell experiment using human brain microvascular endothelial cells (HBMEC), treatment with a nitric oxide synthase (NOS) inhibitor (NOSi) restored decreased nitric oxide (NO) levels.

[0092] Therefore, gintonin can be effectively used for the prevention and treatment of ischemic cerebrovascular disorders, including cerebral infarction, which is expected to provide a market competitive advantage by reducing medical costs required for treatment. [Brief explanation of the drawings]

[0093] [Figure 1] Figure 1 shows the results of evaluating the survival rate after gintonin-induced cerebral infarction modeling (CCAO + NOSi mediated) and showing the effect on the survival curve. [Figure 2]Figure 2 shows the image results of mouse brains extracted and stained with 2,3,5-triphenyltetrazolium chloride (TTC) to investigate the effect of gintonin (GEF) treatment on the volume of cerebral infarction. White indicates the area of ​​cerebral infarction, and red indicates the intact area. [Figure 3] Figure 3 is a graph showing the results of a statistical comparison of the volume of cerebral infarction caused by ischemic stroke based on the image results of mouse brains extracted and stained with 2,3,5-triphenyltetrazolium chloride (TTC) to investigate the effect of gintonin (GEF) treatment on the volume of cerebral infarction. [Figure 4] Figure 4 shows the results of an investigation into the amount of nitric oxide (NO) produced in human brain microvascular endothelial cells (HBMEC), the decrease in nitric oxide production due to treatment with a nitric oxide synthase (NOS) inhibitor (100, 300 μM), and the recovery of nitric oxide production due to pretreatment with gintonin. [Figure 5] Figure 5 shows the results of a reanalysis of the results of treatment with 100 μM of a nitric oxide synthase (NOS) inhibitor and pretreatment with gintonin. Panel A of Figure 5 shows the amount of nitric oxide (NO) produced by HBMEC (human brain microvascular endothelial cells). Panel B of Figure 5 shows the analysis of nitric oxide production converted into relative production levels under different treatment conditions. The results show that gintonin pretreatment restored the nitric oxide levels reduced by 100 μM of the NOS inhibitor (F = 9.819, P value = 0.0019, P value summary = **, significant difference among means (P < 0.05) = Yes, R squared = 0.5670). [Figure 6]Figure 6 shows the results of a reanalysis of the results of 300 μM treatment with a nitric oxide synthase (NOS) inhibitor and pretreatment with gintonin. It was confirmed that the decreased nitric oxide (NO) levels were restored by pretreatment with gintonin, even in the case of treatment with 300 μM of a nitric oxide synthase inhibitor. [Figure 7] Figure 7 shows the results confirming that nitric oxide (NO) levels reduced by 100 and 300 μM nitric oxide synthase (NOS) inhibitors were restored by post-treatment with gintonin. Figure 7A shows the amount of nitric oxide produced by human brain microvascular endothelial cells (HBMEC) under different treatment conditions, the amount of nitric oxide reduced by treatment with the NOS synthase inhibitor (100 and 300 μM), and the amount of nitric oxide restored by post-treatment with gintonin. Figure 7B shows the relative amount of nitric oxide produced under different treatment conditions, confirming that the reduced nitric oxide levels after treatment with the NOS synthase inhibitor (100 and 300 μM) were restored by post-treatment with gintonin. [Figure 8]Figure 8 shows the results of an investigation into the effects of pretreatment with a nitric oxide synthase (NOS) inhibitor followed by treatment with 10 μg / ml gintonin on neuroprotection and injury prevention. This was done to determine whether the functional component of gintonin, LPAR (lysophosphatidic acid receptor), contributes to neuroprotection and injury prevention. Immortalized human brain microvascular endothelial cells (iHBMEC) were pretreated with an LPAR antagonist (Kil6425, 5 μM) for 30 minutes, then treated with 10 μg / ml gintonin as in Example 3 and incubated for 1 hour. After treatment with 100 μM of the synthase inhibitor, the cells were further incubated for 1 hour. We confirmed that gintonin did not restore nitric oxide levels reduced by nitric oxide synthase inhibitor treatment when pretreated with an LPAR antagonist (Ki16425, 5 μM) for 30 minutes (A: F = 6.474, P value = 0.0023, P value summary = **, Significant difference among means (P < 0.05) = Yes, R squared = 0.4473; B: F = 13.89, P value = < 0.0001, P value summary = ****, Significant difference among means (P < 0.05) = Yes, R squared = 0.6345). BEST MODE FOR CARRYING OUT THE INVENTION

[0094] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and that the scope of the present invention is not limited by these examples.

[0095] [Example 1] 1. Preparation of Focal Ischemic Stroke Mouse Model This experiment used C57Bl / 6 male mice (25-28 g), the most widely used animal for stroke experiments worldwide (DBL, Eumseong, Republic of Korea). Ten-week-old mice were kept at least one week prior to the experiment and housed in a vivarium with a regulated temperature (22°C ± 1°C) and humidity (50% ± 10%) under a 12-hour light-dark cycle. Free access to food and water was allowed. All experiments were approved by the Animal Care and Use Committee of Dongguk University Ilsan Hospital.

[0096] C57BL / 6N mice were orally administered saline (n=50), gintonin at 150 mg / kg (n=65), or 300 mg / kg (n=20) once daily for one week. Fifteen randomly selected mice from the 150 mg / kg group were then intraperitoneally injected with an LPAR antagonist (Ki16425, 30 mg / kg) prior to oral gintonin administration. All mice were intraperitoneally injected with a 100 mg / kg NOS antagonist (N-omega-Nitro-L-arginine methyl ester hydrochloride) to induce cerebral autoregulatory dysfunction (CAD). Hemispheric ischemia was then induced via common carotid artery occlusion (CCAO) to produce a mouse model of CCAO-mediated focal ischemic stroke (cerebral infarction).

[0097] [Example 2] Assessment of survival rate after cerebral infarction modeling induced by gintonin in mice We aimed to evaluate the survival rate after gintonin-induced cerebral infarction modeling (CCAO + NOSi mediated).

[0098] Specifically, the survival rates of the saline-treated group (Con) and the gintonin-treated groups (150 mg / kg GEF, 300 mg / kg GEF) prepared in Example 1 were evaluated for 7 days using the Kaplan-Meier method and the log-rank test to obtain the mean survival rate.

[0099] As a result, the group of animals treated with gintonin (150 mg / kg) showed a significant (p=0.009) increase in survival compared to the saline-treated group (p<0.05).

[0100] Furthermore, when the LPA1 / 3 receptor antagonist (Ki16425) was pretreated to block the receptor for lysophosphatidic acids, a substance contained in gintonin, and then gintonin was administered (Ki+150mg / kg), no increased survival effect was observed.Mechanistic studies revealed that lysophosphatidic acid, a substance contained in gintonin, stimulates NOS (nitric oxide synthase) activation, thereby inhibiting CCAO+NOSi-mediated cerebral infarction induction and preventing cerebral infarction (Figure 1).

[0101] [Example 3] Gintonin inhibits CCAO+NOSi-mediated cerebral infarction in mice: preventive effect on cerebral infarction We attempted to confirm the preventive effect of gintonin on cerebral infarction by measuring the area of ​​cerebral infarction after cerebral infarction-induced modeling (mediated by CCAO + NOSi) using gintonin.

[0102] Specifically, the brains of the mouse models prepared in Example 1 were removed, and 2 mm thick brain tissue slices were prepared using a brain matrix. These were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) and photographed with a digital camera. The area of ​​cerebral infarction was then measured from each slice using "Image J" software (the volume of cerebral infarction was calculated by multiplying the area by the thickness).

[0103] Figure 2 shows the images of the TTC staining results, and Figure 3 shows the results of a statistical comparison of the cerebral infarction volumes calculated from the images. In Figure 2, white indicates the cerebral infarction area, and red indicates the uninjured area. In Figure 3, the saline group (Saline, 138.70 ± 16 mm) 3 ) and the 150 mg / kg gintonin-treated group (150 mg / kg GEF, 92.20 ± 14.53 mm 3 When comparing the two groups, we confirmed that the cerebral infarction volume was significantly reduced (approximately 33.57%) in the gintonin-treated group (p=0.005). When the LPA1 / 3 receptor antagonist (Ki16425) was pretreated to inhibit the receptor activity of lysophosphatidic acids, a substance contained in gintonin, and then gintonin was treated (LPAR antagonist + GEF, Ki+150GEF), no preventive effect of gintonin on cerebral infarction was observed. These results suggest that stimulating NOS (nitric oxide synthase) activation by the lysophosphatidic acid component of gintonin may be effective in preventing and treating cerebral infarction (Figures 2 and 3).

[0104] [Example 4] Gintonin exerts neuroprotective and injury-preventive effects on human brain-derived endothelial cells (HBMEC) Based on the in vivo effects of gintonin in [Example 2] and [Example 3], we attempted to confirm whether gintonin also exhibits effects in cell experiments (in vitro).

[0105] First, HBMEC (Human Brain Microvascular Endothelial Cells) or imHBMEC (immortalized Human Brain Microvascular Endothelial Cells) were cultured in appropriate culture medium at 37°C, 5% CO2, and 95% relative humidity. When the cells reached 80-90% confluency (average 2 days), they were pretreated with 10 μg / ml of gintonin and cultured for 1 hour. Next, they were treated with a nitric oxide synthase (NOS) inhibitor and cultured for another hour. The effect of this on nitric oxide (NO) production was then examined. Subsequently, the cells were treated with nitric oxide synthase inhibitors at concentrations of 100 and 300 μM.

[0106] We confirmed that nitric oxide reduction by 100 and 300 μM of the synthase inhibitor was restored by gintonin pretreatment (Figure 4). Figure 4A shows the amount of nitric oxide produced by HBMEC cells under different treatment conditions (DMSO solvent treatment to equalize the effect of the solvent in the treatment reagent, or no treatment (DMSO only)). The amount of nitric oxide reduced by gintonin pretreatment (100 and 300 μM) (eNOS_inh_100, eNOS_inh_300) and the amount of nitric oxide restored by gintonin pretreatment (preGintonin10+eNOS_inh_100, preGintonin10+eNOS_inh_300) are shown. Figure 4B shows the results of an analysis of nitric oxide production under different treatment conditions, converted into relative amounts to account for slight differences in cell characteristics, such as passage and confluency.

[0107] Figure 5 shows a reanalysis of the results obtained by treatment with 100 μM of a nitric oxide synthase inhibitor and pretreatment with gintonin. Figure 5A shows the amount of nitric oxide produced by HBMEC cells per treatment condition, while Figure 5B shows the analysis of nitric oxide production converted to relative levels per treatment condition. In Figure 5B, nitric oxide levels were reduced by approximately 20% by the nitric oxide synthase inhibitor, but were restored by approximately 10–12% by gintonin pretreatment, a recovery of nearly 90% compared to the DMSO vehicle treatment group (no treatment (DMSO only)). This confirms that gintonin has a neuroprotective or preventive effect by restoring nitric oxide levels reduced by treatment with a nitric oxide synthase inhibitor in HBMEC cells. Furthermore, the ANOVA statistical analysis showed a significant p-value of 0.0019.

[0108] Figure 6 shows a reanalysis of the results of 300 μM treatment with a nitric oxide synthase inhibitor and pretreatment with gintonin. Figure 6A shows the amount of nitric oxide produced by HBMEC cells under different treatment conditions, while Figure 6B shows the results of an analysis of nitric oxide production converted into relative production under different treatment conditions. We confirmed that even with 300 μM treatment with a nitric oxide synthase inhibitor, the reduced nitric oxide levels were restored by pretreatment with gintonin.

[0109] [Example 5] Neuroprotective and therapeutic effects of gintonin on human brain-derived endothelial cells (HBMEC) We aimed to determine the neuroprotective and therapeutic effects of gintonin. As in Example 3, HBMEC (Human Brain Microvascular Endothelial Cells) or imHBMEC (immortalized Human Brain Microvascular Endothelial Cells) were cultured in an appropriate culture medium at 37°C, 5% carbon dioxide, and 95% relative humidity in air. When the cells reached 80-90% confluency (average 2 days), they were first pretreated with a nitric oxide synthase (NOS) inhibitor and cultured for 1 hour. Then, they were treated with 10 μg / ml of gintonin and further cultured for 1 hour. The effect of gintonin on nitric oxide (NO) production was investigated to confirm the neuroprotective and therapeutic effects of gintonin.

[0110] We confirmed that the nitric oxide reduction induced by the synthase inhibitors (100 and 300 μM) was also restored by post-treatment with gintonin (Figure 7). Figure 7A shows the nitric oxide production levels in HBMEC cells under different treatment conditions (no treatment (DMSO only)), the nitric oxide reduction (eNOS_inh_100, eNOS_inh_300) induced by synthase inhibitor treatment (100 and 300 μM), and the recovery of nitric oxide after post-treatment with gintonin (preGintonin10+eNOS_inh_100, preGintonin10+eNOS_inh_300). Figure 7B shows the results of analyzing the relative nitric oxide production levels under different treatment conditions.

[0111] As can be seen from the results in Figure 7, the nitric oxide levels decreased after treatment with the synthase inhibitor (100, 300 μM) were also restored by post-treatment with gintonin, confirming that gintonin can have neuroprotective, injury prevention, and therapeutic effects by restoring nitric oxide levels.

[0112] [Example 6] Pretreatment with an LPAR antagonist counteracts gintonin-mediated neuroprotective and injury-preventive effects in human brain-derived endothelial cells (HBMEC) We aimed to determine whether the neuroprotective and injury prevention effects of gintonin (10 μg / ml) treatment after pretreatment with a nitric oxide synthase (NOS) inhibitor were due to the lysophosphatidic acid receptor (LPAR), a functional component of gintonin.

[0113] Specifically, immortalized human brain microvascular endothelial cells (iHBMEC) were cultured in an appropriate culture medium at 37°C, 5% carbon dioxide, and 95% relative humidity. When the cells reached 80-90% confluency (average 2 days), they were first pre-cultured with an LPAR antagonist (Ki16425, 5uM) for 30 minutes, then treated with 10ug / ml of gintonin as described in Example 3. After culturing for 1 hour, they were treated with 100uM of a synthetic enzyme inhibitor and further cultured for 1 hour. The effect on nitric oxide (NO) production was then investigated.

[0114] As a result, we found that gintonin did not restore the nitric oxide levels reduced by nitric oxide synthase inhibitor treatment when pretreated with an LPAR antagonist (Ki16425, 5 μM) for 30 minutes, suggesting that the effect of gintonin was mediated through LPAR inhibition (Figure 8). Figure 8A shows the results of an analysis of the amount of nitric oxide (no treatment (DMSO only)) produced by imHBMEC cells under different treatment conditions, the reduction in nitric oxide (eNOS_inh_100 μM) caused by synthase inhibitor treatment (100 μM), and how the change in nitric oxide production caused by gintonin treatment (preGintonin 10 μg / ml + eNOS_inh_100 μM) was affected by 30 minutes of pretreatment with an LPAR antagonist (Ki16425, 5 μM) (preKi 5 μM + preGintonin 10 μg / ml + eNOS_inh_100 μM). FIG. 8B shows the results of analyzing the relative amount of nitric oxide (NO) produced under different treatment conditions.

[0115] As can be seen from the results in Figure 8, the nitric oxide levels decreased after treatment with a synthase inhibitor (100, 300 μM), but pretreatment with an LPAR antagonist (Ki16425, 5 μM) did not restore the nitric oxide levels decreased by 10 μg / ml of gintonin, suggesting that the effect of gintonin is mediated through LPAR inhibition. Furthermore, the ANOVA statistical analysis results in Figure 8A showed a highly significant p-value of 0.0023, and the results of the analysis of relative nitric oxide production in Figure 8B showed a highly significant p-value of 0.0001. [Industrial Applicability]

[0116] Gintonin, a compound derived from ginseng and containing the LPAR ligand lysophosphatidic acid receptor (LPAR) of the present invention, increased post-stroke survival and reduced infarct volume in a mouse model of focal ischemic stroke. Furthermore, cell experiments using human brain microvascular endothelial cells (HBMEC) confirmed that reduced nitric oxide (NO) levels were restored by treatment with a nitric oxide synthase (NOS) inhibitor (NOSi). Therefore, gintonin may be useful for the prevention and treatment of ischemic cerebrovascular disorders, including cerebral infarction. This is expected to provide a market competitive advantage by reducing medical costs, and thus has industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating ischemic cerebrovascular disease, comprising gintonin.

2. The pharmaceutical composition according to claim 1, wherein the gintonin is a lysophosphatidic acid in the gintonin.

3. 2. The pharmaceutical composition according to claim 1, wherein the ischemic cerebrovascular disorder is ischemic cerebral infarction.

4. The pharmaceutical composition according to claim 1, wherein the ischemic cerebrovascular disorder is caused by a decrease in production of nitric oxide.

5. The pharmaceutical composition according to claim 1, characterized in that the composition reduces the volume of cerebral infarction.

6. The pharmaceutical composition according to claim 1, characterized in that the composition increases the survival rate of individuals who have suffered from cerebral infarction.

7. A health functional food composition containing gintonin for preventing or improving ischemic cerebrovascular disease.

8. A composition for protecting nerve cells, comprising gintonin.

9. The composition according to claim 8, wherein the gintonin is a lysophosphatidic acid in the gintonin.

10. The composition according to claim 8, wherein the nerve cells are nerve cells damaged by a decrease in nitric oxide production.

11. The composition according to claim 8, wherein the nerve cells are cerebrovascular-derived nerve cells.

12. A functional health food composition for protecting nerve cells, comprising gintonin.

13. A method for treating ischemic cerebral disease, comprising administering a composition containing gintonin to a patient suffering from ischemic cerebrovascular disease.

14. Use of gintonin for the treatment of ischemic cerebrovascular disease.

15. Use of gintonin for the manufacture of a therapeutic agent for ischemic cerebrovascular disease.

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