KR20250099037A - Composition for preventing, ameliorating or treating blood clot-related disease or metabolic disease comprising Lycoramine as effective component - Google Patents

Abstract

The present invention relates to a composition containing lycoramine as an active ingredient for preventing, improving or treating a thrombosis-related disease or metabolic disease, wherein lycoramine has the effects of inhibiting platelet aggregation; delaying the time required for carotid artery occlusion; inhibiting neutral fat accumulation and cholesterol accumulation in hepatocytes, reducing the expression amount of genes related to fat synthesis or increasing the expression amount of genes related to fat oxidation; and reducing the content of fat droplets in preadipocytes, reducing the expression amounts of genes related to adipocyte differentiation and genes related to fat synthesis, and increasing the expression amount of genes related to fat decomposition. Therefore, the composition of the present invention containing lycoramine as an active ingredient can be usefully used as a material for a treatment agent for thrombosis-related diseases, a health functional food for improving blood circulation, a treatment agent for metabolic diseases, and a health functional food for improving metabolic diseases.

Description

Composition for preventing, ameliorating or treating blood clot-related disease or metabolic disease comprising Lycoramine as effective component

The present invention relates to a composition for preventing, improving or treating a thrombotic disease or metabolic disease, containing ricoramine as an active ingredient.

Improvement of blood circulation is mainly involved in plasma and blood cells (red blood cells and platelets) that make up blood, which maintain the homeostasis of blood flow and maintain normal hemostasis and protection in damaged or inflamed areas of blood vessels, thereby maintaining the normal function of the human body. However, excessive activation of coagulation factors in the plasma, promotion of platelet aggregation, and abnormality in red blood cell deformability destroy the homeostasis of blood flow, causing cardiovascular diseases such as arteriosclerosis and stroke, which are blood circulation disorders.

In normal blood vessels, homeostasis is maintained by a balance between the activation reaction of the hemostatic mechanism and the inhibitory reaction. However, excessive hemostatic action and the formation of blood clots interfere with the flow of blood, causing blood circulation abnormalities and inducing lesions such as thrombus.

Blood clot-related diseases, which account for approximately 39% of deaths worldwide, have recently been on the rise in Korea due to westernized diets, excessive stress, and an aging population. The global antithrombotic and anticoagulant market size is expected to reach $24.4 billion in 2015, and sales are expected to increase gradually until 2022. In particular, the increase in incidence, prevalence, and number of diagnoses in developing countries such as China and India is expected to contribute to increased sales.

Currently known thrombolytic enzyme agents in use include streptokinase, urokinase, and tissue plasminogen activator (t-PA), which mainly dissolve thrombi indirectly by activating in vivo plasminogen into plasmin.

Streptokinase and urokinase are foreign enzyme substances that convert plasminogen to plasmin, and they have side effects such as fever, allergies, local bleeding, and hypotension. They are also very expensive and classified as pharmaceutical treatments, so they are limited to therapeutics and are not used to prevent thrombosis in the body. Therefore, active research is being conducted on substances that are safe and have excellent blood circulation improvement effects.

Meanwhile, due to recent economic development and changes in eating habits, the incidence of metabolic diseases (metabolic syndromes), including obesity, hyperlipidemia, hypertension, arteriosclerosis, and liver disease, is rapidly increasing. These diseases may occur individually, but they are usually closely related to each other and occur with various symptoms.

Obesity is widely known to cause chronic diseases such as fatty liver, hypertension, diabetes, and cardiovascular disease, and as of 2007, 1.7 billion people, or about 25% of the world's population, were overweight (BMI > 25), 1 in 5 children were obese, and the number of obese children is rapidly increasing, emerging as a serious social problem. Among the obesity treatments sold domestically and internationally, there is 'Xenical', which uses orlistat, which has been approved by the US FDA, as its main ingredient, but Xenical, which inhibits lipase action, has gastrointestinal side effects such as steatorrhea, gas production, and reduced absorption of fat-soluble vitamins.

Dyslipidemia is a condition in which serum lipids are increased or decreased compared to normal. Most cases of dyslipidemia can be caused by causes such as obesity, diabetes, and drinking, but there are also cases in which dyslipidemia is shown due to genetic factors that increase specific lipids in the blood. In addition to dyslipidemia, terms such as hyperlipidemia, hypercholesterolemia, and hypertriglyceridemia are used with similar meanings, but dyslipidemia is a broad disease name that includes all three.

Atherosclerosis is a condition in which fatty substances (plaque) containing cholesterol, phospholipids, calcium, etc. accumulate in the inner lining of blood vessels, reducing the elasticity of the arteries and hardening them, and the narrowing of the arteries causes blood supply to be restricted or pressure to increase, resulting in rupture or detachment.

On the other hand, the liver is an organ that plays a central role in nutrient metabolism, and if liver function is abnormal, problems occur in the body's nutrient metabolism, causing abnormalities in liver functions such as converting glucose into glycogen, converting proteins into albumin, or decomposing unnecessary substances and delivering them to bile. Among them, fatty liver is a disease in which fat such as neutral fat accumulates abnormally in liver cells, causing liver function abnormalities. The initial pathology is simple fatty liver, in which only fat deposition occurs in liver cells, and it is known that the pathology progresses to steatohepatitis and cirrhosis, including liver fibrosis. Fatty liver is classified into alcoholic fatty liver and non-alcoholic fatty liver depending on the cause. Alcoholic fatty liver disease progresses from simple fatty liver disease in the early stage to steatohepatitis and cirrhosis, while non-alcoholic fatty liver disease is thought to remain simple fatty liver and not progress. However, it has been recently reported that there are cases in which non-alcoholic fatty liver disease progresses from simple fatty liver disease to steatohepatitis or cirrhosis.

Most patients with nonalcoholic fatty liver disease have insulin resistance, obesity, diabetes, and hyperlipidemia. In particular, 69-100% of nonalcoholic fatty liver disease patients are obese, and 20-40% of obese patients have nonalcoholic fatty liver disease. In particular, the prevalence of liver disease in male obese patients is higher than in female obese patients. Currently, there are two main types of treatments used for these patients. The first is drugs that treat and improve fatty liver by correcting risk factors, such as anti-obesity drugs, anti-insulin resistance drugs, or anti-hyperlipidemic drugs. The second is drugs that are responsible for the function of restoring damaged liver cells, such as hepatoprotectors, antioxidants, or nutritional support. However, there is currently no effective drug treatment for nonalcoholic fatty liver disease, and only basic treatments such as weight loss through diet therapy and exercise therapy are recommended. In particular, since nonalcoholic steatohepatitis has a high possibility of progressing to cirrhosis or hepatocellular carcinoma, more active drug treatment is essential, and treatments aimed at improving oxidative stress and insulin resistance, which are considered important in the onset and progression of the pathology of nonalcoholic steatohepatitis, are being attempted, but since there is still no treatment with sufficient scientific evidence, the development of highly effective treatments for nonalcoholic fatty liver disease is demanded. Therefore, due to the above demands, there is a need for the production of products that can prevent, improve, or treat metabolic diseases more safely.

Meanwhile, as prior art related to the effect of improving blood circulation or metabolic diseases, Korean Patent No. 2183915 discloses a composition for improving blood circulation comprising a composite extract composed of an extract of Angelica gigas Nakai, an extract of Baeksu-o, and an extract of Ginkgo biloba leaves as an active ingredient, Korean Patent No. 1352591 discloses a composition for improving blood circulation containing a thistle extract, Korean Patent No. 1671248 discloses a composition for preventing or treating non-alcoholic fatty liver and metabolic diseases containing alpha mangosteen as an active ingredient, and Korean Patent No. 1523812 discloses a composition for preventing and treating metabolic diseases containing an extract of Yulcho as an active ingredient, but no composition for preventing, improving, or treating thrombosis-related diseases or metabolic diseases containing the licoramine of the present invention as an active ingredient has been disclosed.

The present invention was derived from the above needs, and provides a composition containing lycoramine as an active ingredient for preventing, improving or treating a thrombosis-related disease or metabolic disease, and completed the present invention by confirming that the lycoramine has the effects of inhibiting platelet aggregation; delaying the time required for carotid artery occlusion; inhibiting neutral fat accumulation and cholesterol accumulation in hepatocytes, reducing the expression amount of genes related to fat synthesis or increasing the expression amount of genes related to fat oxidation; and reducing the content of fat droplets in preadipocytes, reducing the expression amounts of genes related to adipocyte differentiation and genes related to fat synthesis, and increasing the expression amount of genes related to fat decomposition.

In order to solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating a thrombotic disease or metabolic disease containing ricoramine or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for improving blood circulation or preventing or improving metabolic diseases, containing ricoramine or a food-wise acceptable salt thereof as an effective ingredient.

In addition, the present invention provides a feed additive for improving blood circulation or preventing or improving metabolic diseases, containing ricoramine or a food-wise acceptable salt thereof as an effective ingredient.

In addition, the present invention provides a veterinary composition for preventing or treating thrombotic diseases or metabolic diseases, containing ricoramine or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a platelet aggregation inhibitor containing ricoramine or an acceptable salt thereof as an active ingredient.

The present invention relates to a composition containing lycoramine as an active ingredient for preventing, improving or treating a thrombosis-related disease or metabolic disease, wherein lycoramine has the effects of inhibiting platelet aggregation; delaying the time required for carotid artery occlusion; inhibiting neutral fat accumulation and cholesterol accumulation in hepatocytes, reducing the expression amount of genes related to fat synthesis or increasing the expression amount of genes related to fat oxidation; and reducing the content of fat droplets in preadipocytes, reducing the expression amounts of genes related to adipocyte differentiation and genes related to fat synthesis, and increasing the expression amount of genes related to fat decomposition. Therefore, the composition of the present invention containing lycoramine as an active ingredient can be usefully used as a material for a treatment agent for thrombosis-related diseases, a health functional food for improving blood circulation, a treatment agent for metabolic diseases, and a health functional food for improving metabolic diseases.

Figure 1 shows the results of confirming the degree of platelet aggregation according to the lycolamine treatment of the present invention after inducing platelet aggregation by treating collagen. *, **, *** indicate that the degree of platelet aggregation in the lycolamine treatment group was statistically significantly reduced compared to the untreated group (Vehicle) that was not treated with lycolamine. * is p <0.05, ** is p <0.01, and *** is p <0.001.
Figure 2 shows the results of confirming the effect of delaying carotid artery occlusion by treatment with licoramine in an animal model of carotid artery thrombosis. (A) is the result of confirming laser Doppler flow in the carotid artery, and (B) shows the time to carotid artery occlusion according to treatment with licoramine. ### indicates that the time to carotid artery occlusion of the licoramine of the present invention or the positive control group, aspirin (ASA), increased statistically significantly compared to the untreated control group (Vehicle), and p<0.001.
Figure 3 shows the results of confirming the cytotoxicity (A), neutral fat accumulation inhibitory effect (B), and cholesterol accumulation inhibitory effect (C) according to the lycolamine treatment of the present invention in Hepa1c1c cells. Intracellular lipid accumulation was induced by treatment with oleic acid and palmitic acid. ATV is a positive control group treated with atorvastatin. ### means that intracellular neutral fat and cholesterol accumulation statistically significantly increased by oleic acid and palmitic acid treatment, and p<0.001. *, **, *** mean that intracellular neutral fat and cholesterol accumulation statistically significantly decreased by lycolamine treatment, and * means p <0.05, ** means p <0.01, and *** means p <0.001.
Figure 4 shows the results of confirming the gene expression levels of FAS and SREBP1c, which are lipogenic factors, and PPARα and CPT1, which are related to lipogenic oxidation, according to the lycoramine treatment of the present invention in Hepa1c1c cells. ##, ### indicate that the gene expression levels of lipogenic factors (FAS and SREBP1c) in the lipid (0.5 μM oleic acid and 0.25 μM palmitic acid) treatment group (LL) were statistically significantly increased compared to the normal group, or the expression levels of genes related to lipogenic oxidation (PPARα and CPT1) were statistically significantly decreased, and ## is p<0.01 and ### is p<0.001. *, **, *** indicate that the gene expression level of the fat synthesis factor in the ricoramine treatment group of the present invention was statistically significantly decreased compared to the lipid treatment group (LL), or that the expression level of the gene related to fat oxidation was statistically significantly increased. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.
Figure 5 shows the results of confirming the cytotoxicity according to the treatment with ricoramine of the present invention in 3T3L1 cells. ns means there is no statistically significant difference.
Figure 6 shows the results of Oil Red-O staining according to 3T3L1 cell differentiation and lycolamine treatment. (A) is a photograph of Oil Red-O staining, and (B) is a quantitative graph of the results of Oil Red-O staining. ### indicates a statistically significant increase in the fat globule content of the control group in which adipocyte differentiation was induced compared to the normal group (Nor), and p<0.001. *, *** indicate a statistically significant decrease in the fat globule content of the lycolamine treatment group of the present invention compared to the control group in which adipocyte differentiation was induced, and * indicates p<0.05, and *** indicates p<0.001.
Figure 7 shows the results of confirming the changes in the expression levels of adipocyte differentiation-related genes (PPARγ and C/EBPα), lipogenesis-related genes (FAS and ACC), and lipolysis genes (ATGL) according to treatment with licoramine. ### indicates that, compared to the normal group, the expression levels of adipocyte differentiation-related genes and lipogenesis-related genes in the adipocyte differentiation-induced group increased, or the expression levels of lipolysis genes decreased statistically significantly, and p<0.001. *, **, *** indicate that, compared to the adipocyte differentiation-induced group, the expression levels of adipocyte differentiation-related genes and lipogenesis-related genes in the licoramine-treated group of the present invention decreased, or the expression levels of lipolysis genes increased statistically significantly. * is p<0.05, ** is p<0.01, and *** is p<0.001.

In order to achieve the purpose of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating a thrombotic disease or metabolic disease, containing ricoramine or a pharmaceutically acceptable salt thereof as an active ingredient.

Lycoramine has the following chemical formula 1.

In one embodiment of the present invention, the composition has the characteristics of improving blood circulation by inhibiting platelet aggregation due to collagen and reducing the content of neutral fat and cholesterol in the blood.

In one embodiment of the present invention, the composition has the characteristic of improving metabolic diseases by inhibiting fat accumulation and reducing blood triglyceride and cholesterol contents.

The above thrombotic diseases are at least one selected from the group consisting of arterial thrombosis, venous thrombosis, pulmonary embolism, chronic venous ischemia, varicose veins of the lower extremities, deep vein thrombosis, arteriosclerosis, dyslipidemia, cerebral hemorrhage, stroke, and cerebral infarction, but are not limited thereto.

The above metabolic diseases are at least one selected from, but not limited to, dyslipidemia, obesity, hypertension, fatty liver disease, and arteriosclerosis.

The above fatty liver disease is non-alcoholic fatty liver disease, and non-alcoholic fatty liver disease includes, but is not limited to, simple fatty liver, nutritional fatty liver, starvation fatty liver, obesity fatty liver, diabetic fatty liver, steatohepatitis, liver fibrosis, or cirrhosis.

The above salts include common acid addition salts, for example, salts derived from inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid and hydrobromic acid, and salts derived from organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, citric acid, maleic acid, malonic acid, malic acid, tartaric acid, gluconic acid, lactic acid, gestic acid, fumaric acid, lactobionic acid, salicylic acid, phthalic acid, embonic acid, aspartic acid, glutamic acid and acetylsalicylic acid. The above salts also include salts derived from amino acids such as glycine, alanine, valine, isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine and proline. Additionally, the salts include salts of sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.

The composition of the present invention is preferably prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto.

The composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-mentioned effective ingredient, and may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, etc., and such solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, and aerosols, and in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. For parenteral administration, it is desirable to select external skin application or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine epidural, or intracerebrovascular injection methods.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the level of the effective amount can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The composition of the present invention can be used alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention relates to a health functional food composition containing ricoramine or a food-wise acceptable salt thereof as an effective ingredient for improving blood circulation; or preventing or improving metabolic diseases.

In one embodiment of the present invention, the composition has the characteristics of improving blood circulation by inhibiting excessive platelet aggregation and reducing the content of neutral fat and cholesterol in the blood.

In one embodiment of the present invention, the composition has the characteristic of improving metabolic diseases by inhibiting fat accumulation and reducing blood triglyceride and cholesterol contents.

The above composition is preferably prepared in any one dosage form selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the effective ingredient may be added as it is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method. The amount of the effective ingredient may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing a food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the effective ingredient may be used in an amount greater than the above range. There is no particular limitation on the type of the food. Examples of foods to which the effective ingredient may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

When the composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above are 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 a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 g of the composition of the present invention. The composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in mixtures. The proportion of these additives is not particularly important, but the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

In addition, the present invention relates to a feed additive for improving blood circulation or preventing or improving metabolic diseases, containing lycoramine or a food-wise acceptable salt thereof as an effective ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. The term 'feed' in the present invention may mean any natural or artificial diet, meal, etc., or a component of the meal, which is suitable for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feed such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; animal feed such as proteins, inorganic substances, fats, minerals, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

In addition, the present invention relates to a veterinary composition for preventing or treating thrombotic diseases or metabolic diseases, containing ricoramine or a pharmaceutically acceptable salt thereof as an active ingredient.

The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate, ethanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

The veterinary composition according to the present invention may additionally contain fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of a powder, granule, tablet, capsule, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsule, suppository, sterile injectable solution, sterile external preparation, etc.

The effective amount of the veterinary composition according to the present invention can be appropriately selected depending on the individual animal. It can be determined based on factors including the severity of the disease or condition, the sensitivity to the effective ingredient of the present invention according to the age, weight, health status or sex of the individual, the route of administration, the period of administration, other compositions combined or used simultaneously with the composition, and other factors well known in the field of physiology or veterinary medicine.

In addition, the present invention relates to a platelet aggregation inhibitor containing lycoramine or an acceptable salt thereof as an active ingredient. In one embodiment of the present invention, the active ingredient is characterized by having an effect of inhibiting platelet aggregation by collagen.

Hereinafter, the present invention will be described in more detail using examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1. Confirmation of platelet aggregation inhibition effect

Mouse platelets were treated with lycolamine (TRC, L487600, CAS No. 21133-52-8) at various concentrations at 37°C for 15 minutes, and then treated with collagen (3 μg/㎖), a platelet aggregation factor, to activate platelets and induce platelet aggregation. Afterwards, the degree of platelet aggregation was measured and analyzed using a 4-channel platelet lumi-aggregometer (Chronolog Corp, Havertown, PA).

As a result, as disclosed in Fig. 1, the ricoramine of the present invention inhibited platelet aggregation caused by collagen treatment in a concentration-dependent manner.

Example 2. Confirmation of antithrombotic effect in an animal model of carotid artery thrombosis

After anesthetizing the mice with 2% isoflurane, licoramine (1 mg/kg, BW) was intravenously injected (IV) into the left jugular vein of the mice, and the right carotid artery was isolated. Filter paper (2 mm diameter) soaked with FeCl ₃ (10%) was placed on the carotid artery for 2 minutes to induce thrombosis. Thereafter, blood flow was measured using a blood flow meter (AD meter, blood flow meter). At this time, ASA (aspirin, 100 mg/㎖) was administered orally.

As a result, as disclosed in Fig. 2, it was confirmed that the ricoramine of the present invention has an effect of effectively delaying carotid artery occlusion.

Example 3. Confirmation of cytotoxicity, lipid accumulation inhibition effect, and changes in lipid metabolism-related gene expression level according to lycolamine treatment in Hepa1c1c cells

(1) Cytotoxicity evaluation

Hepa1c1c cells were cultured in 96-well cell culture plates at a density of 5 × 10 ⁴ cells/well for 24 h, and then treated with 2.5, 5, 10, and 20 μM lycolamine, respectively. After 24 h, CCK-8 (cell counting Kit-8, Dojindo Molecular Technologies Inc., Rockville, MD, USA) assay was performed. Afterwards, cytotoxicity was confirmed at 450 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

As a result, as disclosed in Fig. 3A, it was confirmed that the ricoramine of the present invention does not exhibit cytotoxicity in Hepa1c1c cells.

(2) Induction of cellular lipid accumulation and measurement of lipid concentration

To induce intracellular lipid accumulation, oleic acid and palmitic acid were dissolved in ethanol to prepare stock solutions with 0.5 mM oleic acid and 0.25 mM palmitic acid, and then conjugated to bovine serum albumin (BSA), respectively. After that, the culture medium was treated with lipids (0.5 μM oleic acid and 0.25 μM palmitic acid), and lycolamine was treated at various concentrations. After culturing for 24 hours, the contents of intracellular cholesterol and neutral fat were measured. At this time, 10 μM atorvastatin (ATV) was used as a positive control.

As a result, as disclosed in Figures 3B and 3C, it was confirmed that the ricoramine of the present invention has an excellent effect in suppressing the accumulation of neutral fat and cholesterol in liver cells.

(3) Confirmation of changes in gene expression related to lipid metabolism

Real-time polymerase chain reaction (real-time PCR) was performed to confirm the regulation of gene expression related to lipid metabolism. Oleic acid and palmitic acid were each conjugated to bovine serum albumin (BSA) in hepatic cell lines, and treated to a concentration of 250 μM to accumulate lipids, and then lycolamine was treated at various concentrations and cultured for 24 h. After washing with PBS solution, RNA was extracted using RNeasy mini kit (QIAGEN). 1 μg of the extracted RNA was quantified using a nanodrop device, and cDNA was synthesized using Maxima first strand cDNA synthesis kit for RT-qPCR (Thermo scientific, Waltham, USA), and then PCR was performed.

Primer sequences of genes related to lipogenesis and lipid oxidation Gene name direction Sequence (5'-> 3') Sequence number Fas Forward ACCTGGTAGACCACTGCATTG 1 Reverse CCTGATGAAACGACACATTCTCA 2 SREBP1c Forward GCAGATTTATTCAGCTTTGC 3 Reverse CCCTACCGGTCTTCTATCAA 4 PPARa Forward GAACAAAGACGGGATGCTGA 5 Forward ACAGAACGGCTTCCTCAGGT 6 CPT1 Forward GTGCAAGCAGCCCGTCTAG 7 Reverse TTGCGGCGATACATGATCAT 8

As a result, it was confirmed that when treated with lycoramine, the expression levels of FAS and SREBP1c genes, which are fat synthesis factors, decreased compared to the untreated control group, and the expression levels of PPARα and CPT1 genes, which are related to fat oxidation, increased (Fig. 4).

Example 4. Confirmation of anti-obesity efficacy of ricoramine in 3T3-L1 cultured cell line

[Cell line culture and differentiation]

3T3-L1 preadipocytes were cultured in high-glucose DMEM containing 10% bovine calf serum (BCS) and 1% penicillin-streptomycin (PEST) at 37°C and 5% _CO2 . For differentiation, 1 × 10 ⁵ cells/well were seeded in 6-well plates, and after 4 days when 100% confluent, differentiation was induced by adding adipogenic cocktail [3-isobutyl-1-methylxanthine (0.25 mM), insulin (1 μg/㎖), dexamethasone (1 μM)] to high-glucose DMEM containing 10% FBS and 1% PEST. During the first 3 days of differentiation, the same culture medium was exchanged every day, and during the middle 3 days of differentiation, the culture medium was exchanged with DMEM containing 10% FBS containing only insulin (5 μg/ml). Afterwards, during the last 3 days of differentiation, the culture medium was exchanged with DMEM containing 10% FBS and used in the experiment. At this time, lycoramine was treated at the same time as the start of differentiation induction according to concentration.

(1) Check cell viability

3T3-L1 cells were differentiated in 24-well plates, treated with lycolamine at various concentrations, and cultured for 48 hours. Afterwards, the absorbance was measured at 450 nm using CCK reagent.

As a result, as disclosed in Fig. 5, it was found that treating 3T3-L1 cells with lycolamine had little effect on cell viability.

(2) Oil Red-O (ORO) dyeing

After washing the differentiated 3T3-L1 cells twice with PBS solution, the PBS solution was completely removed, 10% formalin was added, fixed for 20 minutes at room temperature, and 60% isopropanol was treated and incubated for 5 minutes at room temperature to allow the ORO reagent to be well stained. After that, the plate was completely dried, ORO solution was added, and fat globules were stained for 30 minutes. After staining, the cells were washed several times with distilled water, and the effect of lycolamine on adipocyte differentiation was observed visually using a microscope. To quantify the stained fat globules, 100% isopropanol was added in a well-dried state to elute the ORO staining reagent, and then the absorbance was measured at a wavelength of 490 nm using a spectrophotometer (SpectraMax i3, Molecular devices, CA, USA).

As a result of performing ORO staining after simultaneous treatment with lycolamine and 3T3L1 cell differentiation, it was confirmed that the number of fat globules stained red was significantly reduced in the lycolamine-treated group (Fig. 6).

(3) Measurement and analysis of gene expression level

To confirm the changes in the expression of genes related to fat differentiation and metabolism, real-time polymerase chain reaction (real-time PCR) was performed. RNA was extracted from differentiated 3T3-L1 cells using the RNeasy mini kit (QIAGEN), and 1 μg of RNA was quantified to synthesize cDNA using RT-qPCR (Thermo scientific, Waltham, USA). After that, real-time PCR was performed using the cDNA.

Primer sequences of genes related to fat differentiation and metabolism Gene name direction Sequence (5'-> 3') Sequence number PPARr Forward ACCCTTGCATCCTTCACAAG 9 Reverse AAGAGCTGACCCAATGGTTG 10 C/EBPa Forward GCGCAAGAGCCGAGATAAAG 11 Reverse CGGTCATTGTCACTGGTCAACT 12 FAS Forward ACCTGGTAGACCACTGCATTG 1 Reverse CCTGATGAAACGACACATTCTCA 2 ACC Forward CGCTCAGGTCACCAAAAAGAATG 13 Reverse GGGTCCCGGCCACATAA 14 ATGL Forward CCTCAGGACAGCTCCACCAA 15 Reverse GATTGCGAAGGTTGAACTGGAT 16 GAPDH Forward GAAGGGTGGAGCCAAAAG 17 Reverse GCTGACAATCTTGAGTGAG 18

As a result, treatment with lycolamine decreased the expression levels of PPARγ (peroxisome proliferator-activated receptor gamma) and C/EBPα (CCAAT/enhancer-binding protein α) genes related to adipocyte differentiation, and statistically significantly decreased the expression levels of FAS (fatty acid synthase) and ACC (acetyl-CoA carboxylase) genes, which are fat synthesis-related genes. In addition, it was confirmed that the expression level of ATGL (adipose triglyceride lipase), which is a fat decomposition gene, was statistically significantly increased (Fig. 7).

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Claims (10)

A pharmaceutical composition for the prevention or treatment of thrombotic diseases or metabolic diseases, containing licoramine of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]
A pharmaceutical composition for preventing or treating a thrombosis-related disease or metabolic disease, characterized in that in claim 1, the thrombosis-related disease is any one selected from the group consisting of arterial thrombosis, venous thrombosis, pulmonary embolism, chronic venous ischemia, varicose veins of the lower extremities, deep vein thrombosis, arteriosclerosis, dyslipidemia, cerebral hemorrhage, stroke, and cerebral infarction. A pharmaceutical composition for preventing or treating a thrombosis-related disease or metabolic disease, characterized in that in claim 1, the metabolic disease is any one selected from dyslipidemia, obesity, hypertension, fatty liver disease, and arteriosclerosis. A pharmaceutical composition for preventing or treating a thrombotic disease or metabolic disease, characterized in that in claim 1, in addition to the effective ingredient, it further comprises a pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutical composition for preventing or treating a thrombotic disease or metabolic disease, characterized in that in claim 1, the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. A health functional food composition containing ricoramine or a food-scientifically acceptable salt thereof as an active ingredient for improving blood circulation or preventing or improving metabolic diseases. A health functional food composition for improving blood circulation or preventing or improving metabolic diseases, characterized in that in claim 6, the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage. A feed additive for improving blood circulation, containing ricoramine or a food-based acceptable salt thereof as an active ingredient; or for preventing or improving metabolic diseases. A veterinary composition for the prevention or treatment of thrombotic diseases or metabolic diseases, containing ricoramine or a pharmaceutically acceptable salt thereof as an active ingredient. A platelet aggregation inhibitor containing licoramine or an acceptable salt thereof as an active ingredient.