KR101691855B1 - Pharmaceutical composition comprising the extraction of salicornia spp. as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and health-aid food comprising an extract of Salicornia spp. as an active ingredient for preventing or treating thrombosis. More particularly, the present invention relates to a pharmaceutical composition and health-aid food comprising extract of Salicornia spp. as an active ingredient for preventing or treating/overcoming thrombosis by inhibiting blood coagulation and platelet aggregation. The extract of Salicornia spp. as an active ingredient of a pharmaceutical composition and health-aid food for preventing or treating thrombosis according to the present invention inhibits thrombosis-related enzymes and blood coagulation factors and suppresses aggregation of platelets functioning as initiators of thrombosis, thereby providing strong anti-thrombotic activity. In addition, the extract shows no hemolytic activity to human red blood cells, has excellent thermal stability and causes no loss of an effect of inhibiting blood coagulation factors and thrombosis-related enzymes. Thus, it is expected that the extract can be used for preventing and treating thrombosis, such as ischemic stroke and hemorrhagic stroke, through the improvement of blood circulation. The active ingredient is processed into various forms, such as extract, powder, pills and tablets, so that it may be administered any time. Thus, the present invention is useful for pharmaceutical industry and food industry.

Description

[0001] PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACTION OF SALICORNIA SPP. [0002] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing Salicornia spp. As an active ingredient, and more particularly, To a pharmaceutical composition for preventing or treating / improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation and a health functional food.

As a constituent of human body, blood has various important functions such as oxygen and nutrients, the function and buffering function of waste products, maintenance of body temperature, control of osmotic pressure and maintenance of ion balance, maintenance of moisture, regulation of fluidity, maintenance and regulation of blood pressure, have. Normal blood circulation facilitates blood circulation by complementary regulation of the blood coagulation system and thrombolysis system in the body. Among them, the mechanism of the blood coagulation system is that the platelets adhere to the blood vessel walls and coagulate to form platelet thrombus , The blood coagulation system is activated and fibrin thrombi are formed centering on platelet aggregation mass.

On the other hand, the production of fibrin thrombus is activated by thrombin involved in fibrin clotting through several steps of a number of blood coagulation factors to finally produce a fibrin monomer from fibrinogen. The fibrin monomers are polymerized by calcium, Cells to form a cross-linked fibrin polymer by factor XIII and produce a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation by activating platelet, V factor, and Factor VII to promote blood coagulation. Therefore, the activity inhibitor of thrombin can be used as a prophylactic and therapeutic agent very useful for various thrombotic diseases caused by excessive blood coagulation. On the other hand, prothrombin activation after sequential activation of factor XII, factor XI, factor IX and factor X is known to activate thrombin in the endogenous thrombogenesis pathway, so that specific inhibition of blood coagulation factors is also important. It is becoming a target. To date, various anticoagulants such as heparin, coumarin, aspirin, and europaine have been used for the prevention and treatment of thrombotic diseases. However, these anticoagulants are not only very high in price, but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

Salicornia spp. Is called as 퉁 퉁 퉁 마 디 하여 하여 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 퉁 Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal Sal It is called "Hamcho". In other countries, it is called Crabgrass, Slander glasswort, Glassworts, Samphire. It has a moisture content of 90.9%, a ash content of 4.6 to 6.2%, and ash content in the wastewater, which is rich in various minerals and enzymes contained in seawater while growing on land. It contains about 70% of salt. In addition, amino acids such as glutamic acid, aspartic acid, lysine and tyrosine are contained. Minerals include sodium, potassium, calcium, magnesium, zinc, iron, copper, manganese and the like. And has been recognized as a nutritionally important edible plant. In Korea, it grows mainly on the west coast and Ulleungdo. The stem has many nodes and strikes. The branch is divided 2 ~ 3 times and faces each other. As it grows, it becomes fleshy, thick and dark green. Fruits ripen round and flat at the end of September to mid-October. In autumn when the growth is complete, the stems and branches turn from green to red. About 60 varieties are known worldwide, and they are attracting attention as a food having a unique salty taste or being widely used in medicines, textiles, beer manufacturing, and chemical industries and having important economic value. In many parts of the world, it is used as food for salads and food, for extraction oil or for livestock.

It is known that Tungtung maddy is effective not only for food but also for medicinal use in the private sector and is effective for cancer, sinusitis, arthritis, hypertension, back pain, obesity, hemorrhoids, diabetes, thyroiditis, asthma, constipation bronchitis and liver disease Jo, YC et al., 2002. Studies of pharmacological effects of glasswort (Salicornia herbacea L.) Kor J. Medicinal Crop Sci. 10: 93-99). In fact, it has more than 50% dietary fiber, various seaweed polysaccharides, essential amino acids, essential fatty acids and crude saponin, and it is effective to eliminate constipation and eliminate constipation, and also to treat obesity by decomposing neutral fat And is known to be effective in enhancing immunity, inhibiting cancer cell growth, hyperlipidemia, hyperglycemia, and hypertension. In addition, it has been known as a health food and health functional food material because it has various effects such as softening skin, enhancing gastrointestinal function, bronchial asthma and bronchitis, and reducing blood sugar level of diabetes.

In the meantime, the academic researches on the antimicrobial substance of mungbean were as follows: Analysis of physicochemical components of green tea (Shin et al., 2002, Korean J. Plant Res. 15: 216-220), strong antioxidant activity in ethyl acetate fraction and butanol fraction of methanol extract of green tea Inhibition of NO production and inhibition of iNOS and IL-1beta cytokine expression in the ethyl acetate fraction of the active and methanol extracts are known (Lee, Man-Hwi, 2007, Inhibitory effect of RGS-2 knockout mice , 2006, Int Immunopharmacol. 6, 1451-1458), prevention of hyperlipidemia and inhibition of hypertension (Hwang JY et al., 2007, Nutr Res Pract. (Karadeniz et al., 2014, Mar Drugs, 12, 5132-5147), etc. have been reported in the treatment of osteoporosis (Park, SH, et al., 2006, Arch Pharm Res. 29, 256-264) . Inhibitory effects of green tea on anti-obesity and hepatic accumulation (Pichiah & Cha, 2014, J Sci Food Agric., Salicornia herbacea for weight gain and hepatic lipid accumulation in obese ICR mice fed a high-fat diet) (Sung JH et al., 2009. Biosci Biotechnol Biochem. 73, 552-556), antioxidant and cancer cell growth inhibitory activity (Sung JH et al., 2009. Chem Biol Interact 181, 366-762) 1982, 200-209, < RTI ID = 0.0 > Hwang YP et al., 2010. Toxicol Lett. 198, 200-209, < Ryu DS et al., 2009, J Microbiol Biotechnol. 19, 1482-1489). Recently, vinegar production using antioxidant and anti-fatigue antioxidants has been reported (Cho, HD et al., 2015, J Sci Food Agric. Production of novel vinegar having antioxidant and anti-fatigue activities from Salicornia herbacea L.) In addition, the nitrite scavenging activity and the acetylcholine ester inhibition effect of the seeds of Hamcho were also reported (Kim, et al., 2013. Korean Society for Biotechnology and Bioengineering Journal 28 (6): 372-379). Recently, it has been reported that weakly coagulase (thrombin) inhibition and blood coagulation factor inhibition are inhibited in the green tea extract treated with viscozyme and celluclast enzyme at 50 ° C after drying desiccated green tea (Jang HS et al., 2007. Annals of Nutrition & Metabolism, 51: 119-125).

In addition, patents relating to the Tungtung Mardi are mostly patents related to the method of producing vegetable salt, and the method of producing vegetable salt and the method of producing vegetable salt [patent registration No. 10-0448673, patent registration No. 10-0448674], production of liquid vegetable salt Method and liquid vegetable salt [Patent Registration No. 10-0448672], a salt substitute derived from Tungtung Mardi, a method for producing the salt substitute [Patent Registration No. 10-0784229], and a method for manufacturing a Hanshu Salt Salt [Patent Registration No. 10-0587713] It is known. Patent patents related to processed products using the tungsten noodle include a tea composition containing tungsten noodles and a method of producing the same [Patent Registration No. 10-0402695], beverages containing tungsten noodle extract [Patent Registration No. 10-0441111] [Patent Registration No. 10-0506794], a leaching tea containing a herbal ingredient and a method for producing the same, [Patent Registration No. 10-0556085], a functional Kim using charcoal, [Patent Registration No. 10-0595749], Functional Kim containing Hamcho and Cordycepsum and its production method [Patent Registration No. 10-0824018], Seasoned Kimchi using green tea and its production method [Patent Registration No. 10-0736834] Kim, and a method for producing the same [Patent Registration No. 10-0470191] Liquid composition for beverage containing extract of green tea [Patent No. 10-0724705], cosmetic composition containing green tea [Patent Registration No. 10-0362498 , Kimchi containing green tea and its manufacturing method [Patent 10-0422213], Functional beverage using green tea and its production method Functional health food and manufacturing method thereof [Patent registration No. 10-0659015], Manufacturing method of Hamcho samgyetang [Patent registration No. 10-0759315], Green tea A method for producing a mixed cosmetic pack [Patent Registration No. 10-0538536], a manufacturing method of Hamcho Ogoki [Patent Registration No. 10-0539104], a process for producing an edible plant fermented product, an edible plant fermented product produced thereby, [Patent Registration No. 10-0891608], a method for producing a fermented beverage for diet using hamcho juice as a main ingredient [Patent Registration No. 10-062328], a method for producing a cucumber containing green tea [Patent No. 10-0857015 [Patent No. 10-0814443], a method for producing coated rice using a green tea extract and alginic acid [Patent Registration No. 10-0793826], a natural seasoning containing green tea Leaf cake composition and method for producing the same [Patent Registration No. 10-0770763 [Patent No. 10-0775927], a method for producing green tea pickled pickled jam and a green tea pickled pickled jam made by the same method [Patent Registration No. 10-0733585], and the like. The most recent patents related to fermented green tea are lactic acid fermented green tea using lactic acid bacteria and its preparation method [patent registration 10-1118678], manufacturing method of fermented fermented product [patent registration 10-1157648], fermented microorganism using fermented meat and fermentation A method of producing food [Japanese Patent Laid-Open No. 10-2011-0127765] and the like are known.

On the other hand, the patents relating to the useful physiological activity of the Tungtung section include a novel compound having antioxidative activity isolated from Tungtung mardi and a method for its production [Patent Registration No. 10-0501833], 3-caffeoyl- A composition for prevention and treatment of gout containing hydrocaffeoyl-quinic acid [Patent Registration No. 10-0569244], a composition for prevention and treatment of obesity containing 3-caffeoyl-4-dihydrocappahein quinic acid 10-0759466], a composition for treating myeloid leukemia and immunity enhancement comprising a green tea extract [Patent Registration No. 10-0510174], a composition for a rectal preparation using green tea [Patent Registration No. 10-0495767] A composition for treating and preventing blood sugar lowering and obesity including an extract mixture of green tea (Patent Registration No. 10-0610565) is known.

However, there is no known patent for an active extract of Toyochunnamidis which shows strong antithrombotic activity through inhibition of blood coagulation and inhibition of platelet aggregation so far, and which is not toxic.

KR 10-2011-0081007 A

 Jang HS et al., 2007. Annals of Nutrition & Metabolism, 51: 119-125.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the problems of the prior art as described above, and it is an object of the present invention to provide a pharmaceutical composition for preventing or treating thrombotic diseases, .

In order to solve the above problems, the present invention provides a pharmaceutical composition for prevention or treatment of thrombosis, which comprises Salicornia spp. Extract as an active ingredient.

Preferably, the extract is a hot-water extract or an ethanol extract of Tochigi herb.

Preferably, the extract is a ethyl acetate fraction.

The ethyl acetate fraction is preferably a 10-50% methanol solubility material obtained from the ethyl acetate fraction.

The present invention also provides a health functional food for preventing or ameliorating a thrombotic disease containing Salicornia spp. Extract as an active ingredient.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the extract of Tungtung Co., Ltd. as an active ingredient of the health functional food inhibit the coagulation inhibition effect of platelets which inhibits thrombogenesis-related enzymes and blood coagulation factors and plays a role of initiating thrombus formation And exhibits excellent hemostatic activity against human erythrocytes, exhibits excellent thermal stability, and is in a condition of acidic pH 2 and loss of blood coagulation factor inhibitory effect and thrombogenic enzyme inhibitory effect even in plasma It is expected that it can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation and the active ingredient can be processed into various forms such as extract, powder, ring and tablet, It can be used in pharmaceutical industry and It is a very useful invention in the food industry.

FIG. 1 shows human platelet aggregation inhibitory activity of organic solvent sequential fractions of the extract of Tungstenhodi hot water extract: 1: solvent control (DMSO), 2: aspirin (0.5 mg / ml), 3: aspirin (0.25 mg / (0.25 mg / ml), 5: ethyl acetate fraction (0.25 mg / ml) of hot water extract of Tungtung Mardi, 6: butanol fraction (0.25 mg / ml) of hot water extract of Tungtung Mardi, 7: Water residue (0.25 mg / ml) after the organic solvent fraction of the hot-water extract.
FIG. 2 shows human platelet aggregation inhibitory activity of the fraction separated by methanol concentration in the ethyl acetate fraction of the extract of Tungtung Nodi hot water extract: 1: solvent control (DMSO), 2: aspirin (0.25 mg / ml) 10% methanol soluble fraction (0.25 mg / ml) of the ethyl acetate fraction, 4: 10% methanol soluble fraction (0.15 mg / ml) of the antithrombotic ethyl acetate fraction, 5: 30% methanol soluble fraction 50% methanol soluble fraction (0.25 mg / ml) of the anti-thrombotic ethyl acetate fraction, 8: 30% methanol soluble fraction of the anti-thrombotic ethyl acetate fraction (0.15 mg / 50% methanol soluble fraction of ethyl acetate fraction (0.2 mg / ml).

Hereinafter, the present invention will be described in detail.

The inventors of the present invention collected the extract of Tungtung nodule as an antithrombotic active ingredient in a certain way in order to test the antithrombotic effect on the anthelmintic nodule. The extract showed no hemolytic activity on human erythrocytes, And acid stability. Thus, the extract was used as a pharmaceutical composition for the prevention or treatment / improvement of thrombosis and as a health functional food.

Specifically, to develop a pharmaceutical composition and a health functional food for the prevention or treatment / improvement of thrombosis using a microorganism known to be excellent in the blood-improving effect in the private sector, Extracts and ethanol extracts were separately prepared and their antithrombotic activity was compared with that of thrombin time, prothrombin time and activated partial thromboplastin time for human plasma and human thrombin, aPTT), and it was confirmed that they had good anticoagulant activity, and that they did not show hemolytic activity on human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which comprises Salicornia spp. Extract as an active ingredient.

Preferably, the extract is a hot-water extract or an ethanol extract of Tochigi herb.

Preferably, the extract is a ethyl acetate fraction.

The ethyl acetate fraction is preferably a 10-50% methanol solubility material obtained from the ethyl acetate fraction.

The present invention also provides a health functional food for preventing or ameliorating a thrombotic disease containing Salicornia spp. Extract as an active ingredient.

Hereinafter, the extract of Toyochimono nodule according to the present invention, the method for producing each active fraction material, the efficacy experiment and the like will be described in more detail.

The present invention relates to a method for preparing an extract, Preparing sequential organic solvent fractions of hexane, ethyl acetate, and butanol from the extract of Tungsten manganese and preparing a water residue obtained therefrom; The ethyl acetate fraction was dissolved in a methanol solution (10, 30, 50, 70, 100% (v / v) methanol solution) of various concentrations in succession to evaluate the antithrombotic activity of the extract and the fraction. Methanol solubility Recovering the material and examining the stability of the ethyl acetate fraction and the respective concentration of the methanol soluble material.

In the composition of the present invention, the "extract of Tungtung nodule" is obtained by collecting and washing the mature outstock of Tungtung mound from the beginning of September to the beginning of October, extracting it with an organic solvent, Filtration using a filter net, and concentration under reduced pressure.

The organic solvent used in the present invention may be any organic solvent such as water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol (methanol, ethanol, alcohol, propanol, butanol etc.) having 1 to 4 carbon atoms, a mixed solvent of the lower alcohol and water , And hot water, 95% ethanol or ethyl acetate extraction is most preferred.

As a preferred embodiment, the extract of Toyochu-mushroom of the present invention can be used by extracting it with hot water, ethanol or ethyl acetate. Here, the hydrothermal or ethanol extract may be sequentially or separately fractionated with an organic solvent of hexene, ethyl acetate and butanol to obtain a hexane fraction, an ethyl acetate fraction and a butanol fraction and a water residue, respectively.

In the present invention, thrombin time, prothrombin time, apathy time, and human platelet aggregation inhibitory activity were measured at a concentration of 5 mg / ml of extract of Toyochu-mugi, showing increased activity compared to no-added sphere.

In addition, the ethylacetate fraction of the hydrothermal extract of Tungtung Mardi strongly inhibited human platelet aggregation while significantly prolonging thrombin time, prothrombin time, and apathy time at a concentration of 5 mg / ml. This means that the ethylacetate fraction of the hot-water extract of Tungtung-madi has very strong antithrombotic activity.

In order to remove the hemolytic active substance from the ethyl acetate fraction, the methanol solution of various concentrations was sequentially treated. As a result, the thrombin time, prothrombin time extension, and prothrombin time were increased at 5 mg / ml for 30-50% methanol- It was confirmed that the 30-50% methanol solubility material can be used as an actual antithrombotic agent. Considering the prothrombin time, prothrombin time, and apathy time at the 1.5 mg / ml added concentration of aspirin (Protect), a commercially available anti-thrombotic agent, 1.99 times, 1.35 times, and 1.77 times longer than the no- The 30-50% methanol solubility product obtained from the ethyl acetate fraction of the extract of Tungtung-madi hydrothermal extract suggests that it can replace anticoagulants such as aspirin, which have high adverse effects.

On the other hand, the 10% methanol soluble substance of the ethyl acetate fraction of the extract of Tungtung nodule had a very weak blood coagulation inhibitory activity and a very strong platelet aggregation inhibitory activity. Actually, platelet aggregation inhibitory activity was stronger than that of 30 ~ 50% methanol solubility substance, and almost same coagulation inhibition activity as as antiplatelet aspirin was observed. The 10% methanol soluble substance of the ethyl acetate fraction of Tungtungmadi hot water extract was used as a platelet aggregation inhibitor It is confirmed that it is available.

The extract of Tungtung nodule and the active fraction thereof of the present invention can be prepared into powders through a conventional powdering process such as vacuum distillation and freeze drying or spray drying. They are not degraded by various degradation enzymes in the plasma, and maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. Such diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, , Deep vein thrombosis, lower limb edema, pain and acute peripheral artery occlusion. Vein thrombosis can include deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, injections of sterilized injection solutions And may be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

In a preferred embodiment, the solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, for example starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Perfumes, preservatives, and the like.

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

As a preferable example, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight per day Or every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell.

In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , Preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be variously used for foods and beverages effective for prevention or improvement of thrombosis. Examples of foods containing the active ingredient of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage .

The active ingredient of the present invention may generally be added in an amount of 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention may contain, as an additional ingredient, a food-acceptable food-aid additive such as natural carbohydrates and various flavors, in addition to containing the above-mentioned compound as an essential ingredient in the indicated ratio.

Examples of the natural carbohydrate include sugar sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

Examples of the flavoring agent include tau martin; Natural flavoring agents such as stevia such as rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail by way of examples. The following examples are only exemplary embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[ Example ]

Example  1: Manufacture of tungsten carbide stem and branch extracts and their Anti-thrombosis  Activity evaluation

In September 2014, Salicornia Herbacea was divided into stem and branches. 10 times of ethanol was added to each sample, and the extract was repeated twice at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to prepare ethanol extracts. In the case of the hot-water extract, 10 times of distilled water was added to each of the stem and branch, and the mixture was heated at 100 ° C for 1 hour and then cooled. After repeating the above procedure once, the extract was collected and filtered, To prepare hot water extract. The extraction efficiency of each site and the antithrombotic activity of the extract were evaluated, and the results are shown in Tables 1 and 2.

In this study, anticoagulant activity was evaluated as an antithrombotic activity of Toxemus sinensis extract (Sohn et al., 2004. Kor J. Pharmacogn 35, 52-61 ; Kwon et al., 2004. J. Life Science, 14. 509-513; Li et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and apathy time. Plasma was obtained from a commercial control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and thrombin time, prothrombin time and apathy measurement were performed as follows.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl 2 and 10 μl of sample extract at various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C. and 100 μl of plasma was added And the time until the plasma coagulated was measured. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 32.1 sec. The thrombin inhibitory effect was expressed as the average value of the experiments repeated three or more times. The thrombin inhibitory activity was expressed by the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

Prothrombin time ( prothrombin  time)

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution to tubes of Amelung coagulometer KC-1A (Japan), heat at 37 ° C for 3 minutes, add 130 μl of PT reagent, The time from the start of the experiment to the start of the experiment was expressed as the average value of the experiments repeated three times. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 18.1 sec. The prothrombin inhibitory activity was expressed as the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

aPTT  (activated Partial Thromboplastin  Time)

Add 50 μl of aPTT reagent (Sigma, ALEXIN ^™ ) to the tubes of Amelung coagulometer KC-1A (Japan) and incubate for 3 minutes at 37 ° C. Add 100 μl of plasma and 10 μl of various concentrations of sample extract Min. Then, 50 μl CaCl 2 (35 mM) was added and the time until the plasma coagulated was measured. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 55.1 seconds. The results of aPTT were expressed as the mean value of three repeated experiments, and the coagulation factor inhibitory activity was expressed as aPTT time divided by the aPTT time of the solvent control.

[Table 1] By extraction solvent  Extraction efficiency and anticoagulant activity of extract

As shown in Table 1, the hydrothermal extract and ethanol extract of Tungtung nodule showed anticoagulant activity but were weak compared with aspirin. In particular, ethanol extracts from the extracts of the stem extracts showed better anticoagulant activity than the extracts from the stem extracts, especially the ethanol extracts from the hot - water extracts. In particular, the ethanol extracts from the tungstrum stem showed thrombin time and prolonged prolongation Respectively.

Meanwhile, platelet aggregation inhibitory activity was evaluated as an antithrombotic activity evaluation. Platelets are cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentration instead of nucleus, and circulating blood vessels together with various blood cells. Is an important cell that secretes factors and initiates thrombogenesis by forming a primary hemostatic plug in association with collagen exposed by damage of endothelial cells. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombogenesis to be. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity (Platelet aggregation inhibition activity)

Human platelets were used as platelets and washed once with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, the cells were resuspended in a suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 0.49 mM MgCl ₂ , 0.25% gelatin, pH 7.4) and incubated at 3,000 rpm for 10 minutes After centrifugation, the cells were resuspended in a suspending buffer and the platelet count was adjusted to 4 × ^{10 9} / ml. Then platelet aggregation was measured at 37 ° C using a whole-blood agarometer (Chrono-log, USA) after 2.5 μl of collagen was added to 1 ml of suspension and reacted for 5 minutes.

[Table 2] By extraction solvent  Platelet Aggregation Inhibitory Activity of Extracts

As shown in Table 2, the hydrothermal and ethanol extracts of Trichoderma nodule stems and branches showed little inhibitory activity on human platelet aggregation.

Example  2: Sequential organic solvent of extract of Tungtung nodule Fraction  Preparation and analysis of their components

Based on the antithrombotic effect of the extract of Tungtung nodule obtained in Example 1, the extract of Tungtung maddy was prepared in consideration of economy of mass productivity, process safety, and the like. The hot-water extract was prepared in the same manner as in Example 1, and the extract was fractionated with hexane, ethyl acetate and butanol sequentially, and finally, The water residue was collected. Table 3 shows the extraction efficiency of the bulk hot water extraction, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction. Total polyphenol, total flavonoid, total sugar, and reducing sugar content were determined by the analysis of components of hot water extract and fractions. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of saturated Na ₂ CO ₃ solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. The total flavonoid content of each sample was measured by stirring for 18 hours in methanol. To the 400 μl of the filtered extract, 4 ml of 90% diethylene glycol was added, 40 μl of 1 N NaOH was added, and the absorbance at 420 nm was measured at 37 ° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 3] Extracts and their sequential Fraction  Comparison of yield and component analysis

As shown in Table 3, 68.1 g of hot water extract was obtained from 1 kg of Tungtung nodule outpost. As a result, 39.9% of the hot water extract was converted into butanol fraction and 54.8% of the hot water extract was converted to a water residue. The ethyl acetate fraction recovered 4.7%, which means that 3.2 g of the ethyl acetate fraction can be recovered from 1 kg of Tungtung nodule. On the other hand, less than 0.01% of the hexane fraction was recovered and excluded from the experiment in the future.

The total polyphenol contents of the ethyl acetate fraction were more than 10.4 times higher than that of the hot water extract, and 254.8 mg / g of the polyphenol component was concentrated. Respectively. Ethyl acetate fraction showed the highest value of 63.9 mg / g in total analysis, and about 50% of the ethyl acetate fraction was identified as reducing sugar. Therefore, it is expected that the ethyl acetate fraction of the extract of Tungtung nodule will exhibit various physiological activities. In fact, the ethyl acetate fraction exhibits a strong antioxidative activity and will additionally contribute to the inhibition of carcinogenesis, the inhibition of aging, It is expected.

Example  3: Extract and sequential extract Organic solvent Fraction  Evaluation of blood coagulation inhibitory activity

The anti-thrombocyte activity of the extract of Tungtung-madi hot water and its organic solvent fraction was evaluated in the same manner as in Example 1, and the results are shown in Table 4.

[Table 4] Heat number  Extracts and Fraction Anti-thrombosis  activation

As shown in Table 4, when aspirin was eluted at a concentration of 1.5 mg / ml, the prothrombin time, the prothrombin time, and the apathy time were extended by 1.76, 1.48, and 1.59 times, respectively. On the other hand, the hydrothermal extract of Tungtung Mardi showed no antithrombotic activity up to a concentration of 6 mg / ml and showed thrombin inhibitory activity at a concentration of 7 mg / ml. In addition, antithrombotic activity was not observed in the butanol fraction and water residue of the hot - water extract. However, the ethyl acetate fraction showed the same thrombin inhibition as aspirin and prodrombin inhibition (prothrombin time) and blood clotting inhibition (apathy time) stronger than aspirin at 5 mg / ml concentration. As a result, the ethyl acetate fraction, which is a medicinal product of the extract of Tungtung nodule extract, is expected to be able to replace aspirin, which is a conventional antithrombotic agent, exhibiting a side effect such as gastrointestinal disorder due to a very strong antithrombotic activity. It would be possible to develop a commercial antithrombotic agent.

Example  4: Extract and sequential Organic solvent Fraction  Evaluation of human platelet aggregation inhibitory activity

The platelet aggregation inhibitory activity was evaluated in the same manner as in Example 1 as an evaluation of the antithrombotic activity of the extract of Tungtung Mardi and the organic solvent fraction thereof. The results are shown in Table 5 and FIG.

[Table 5] Heat number  Extracts and Fraction  Human platelet aggregation inhibitory activity

As shown in Table 5 and FIG. 1, aspirin used in clinical practice as a platelet aggregation inhibitor inhibited platelet aggregation in a concentration-dependent manner, and exhibited about 50% aggregation inhibition at an aspirin concentration of 0.25 mg / ml under the above experimental conditions. On the other hand, the extracts and water residues of Tungtungmodi showed 207.5% and 183.8% aggregation of platelets at the concentration of 0.25 mg / ml, which promoted platelet aggregation. The butanol fraction of the hot - water extract showed slight promoting activity on the platelet aggregation, but the ethyl acetate fraction exhibited 63.9% agglutination at 0.25 mg / ml concentration, indicating strong agglutination inhibition. This result shows that the ethyl acetate fraction of the extract of Tungtung-madi hydrothermal extract is an anti-platelet agent capable of replacing aspirin, which is a side effect such as gastrointestinal disorder, since the ethyl acetate fraction exhibits platelet aggregation inhibitory activity comparable to aspirin even in the pure water- As shown in FIG.

Example  5: Extract and extract Fraction  Human erythrocyte hemolytic activity

It is an edible plant that is registered as a food ingredient and secured. In order to evaluate the possibility of acute toxicity of the extract of Dongchungmodi hot water and fractions thereof, human hemolytic hemolytic activity was evaluated and the results are shown in Table 6. The hemolytic activity was assessed in accordance with the existing reports (Jung In-chang, Son Ho Yong, 2014 ㆍ Korean J. Microbiol. Biotechnol. 42: 285-292). In brief, 100 μl of human erythrocytes washed three times with PBS were diluted in 96-well microplates , 100 μl of various concentrations of sample solution was added and the reaction was allowed to proceed at 37 ° C for 30 minutes. Then, the reaction solution was centrifuged for 10 minutes at 1,500 rpm, and 100 μl of the supernatant was transferred to a new microtiter plate. The hemoglobin efflux 414 nm. Triton X-100 (1 mg / ml) was used as an experimental control for erythrocyte hemolysis. DMSO (2%) was used as a solvent control of the sample. Hemolytic activity was calculated using the following formula.

[Table 6] Heat number  Extract and its Fraction  Human erythrocyte hemolytic activity

First, DMSO and water used as controls did not have hemolytic activity, and triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. On the other hand, it was confirmed that the hot water extract, butanol fraction and water residue of Tungtung Mardi showed no red blood cell hemolysis phenomenon up to a concentration of 0.5 mg / ml and thus did not have acute toxicity and erythrocyte hemolytic activity. However, the ethyl acetate fraction having excellent antithrombotic activity exhibits a hemolytic activity against human erythrocytes in a concentration-dependent manner, and it has been confirmed that there is a possibility that practical use may be restricted. Therefore, a method for easily removing human erythro hemolysin from the ethyl acetate fraction while maintaining strong antithrombotic activity has been sought through the following examples.

Example  6: Tongtungmadi Anti-thrombosis  Active ethyl acetate Fraction  Modulators and their human hemolytic hemolytic activity

The antithrombotic active ethyl acetate fraction of Tungtung Mardi was fractionated sequentially with 10, 30, 50, 70, and 100% (v / v) methanol to recover the solubilized material in each solvent condition. The recovery rates at each fraction and their human erythrocyte hemolytic activity are shown in Table 7.

[Table 7] Ethyl acetate Fraction Purification yield and  These human hemolytic hemolytic activity

As a result, the strong hemolytic activity of human erythrocyte in the ethyl acetate fraction was converted into 70% methanol solution, and the sequential fraction of 10, 30, and 50% methanol solution showed no hemolytic activity. Therefore, it was confirmed that the lipophilic component in the ethyl acetate fraction was related to the erythrocyte hemolytic activity.

Example  7: Tongtungmadi Anti-thrombosis  Active ethyl acetate Fraction  Anticoagulant activity of 30 ~ 50% methanol soluble substance

The anticoagulant activities of the 10%, 30%, and 50% methanol fractions without human hemolytic hemolytic activity of Example 6 are shown in Table 8.

[Table 8] Anti-thrombosis  activation Purified water  Anticoagulant activity

Aspirin showed anticoagulant activity in a dose dependent manner. At 5.0 mg / ml, thrombin time, prothrombin time, and apathy time were all extended more than 15 times as compared with no. However, severe gastrointestinal disorders such as aspirin is difficult to use at such high concentrations, usually 100mg a day will be taken. On the other hand, the fraction of 30% and 50% methanol soluble substance obtained from the ethyl acetate fraction of the hot-water extract showed an extended thrombin time of 15 times or more at the concentration of 5 mg / ml. Particularly, the 50% methanol fraction had not only thrombin time, Prothrombin time and apathy time were also prolonged by at least 15 times. Therefore, it was confirmed that the 30% to 50% methanol soluble fraction of the ethyl acetate fraction of the hot water extract of Tungtungmadi showed strong anticoagulant activity without hemolysis of human erythrocytes.

Example  8: Tongtungmadi Anti-thrombosis  Active ethyl acetate Fraction  Platelet Aggregation Inhibitory Activity of 10 ~ 50% Methanol Soluble Substance

The results of evaluating the human platelet aggregation inhibitory activity of the 10%, 30%, and 50% methanol fractions without human hemolytic hemolytic activity of Example 6 are shown in Table 9 and FIG.

[Table 9] Anti-thrombosis  activation Purified water  Human platelet aggregation inhibitory activity

As shown in Table 9 and FIG. 2, aspirin, which is an antiplatelet agent used in clinical practice, was found to be a highly potent platelet aggregation inhibitor at a concentration of 112.96 μg / ml required for 50% inhibition of platelet aggregation under the experimental conditions. 10% methanol solubility, 30% methanol solubility, and 50% methanol solubility were also found to be very potent platelet aggregation inhibitors. Considering the potent anticoagulant activity of the 30% methanol solubility material and the 50% methanol solubility material of Example 7, the 30-50% methanol solubility material from Tungtung Mardi, without acute toxicity, inhibits blood coagulation-related enzymes and coagulation factors , And exhibited excellent antithrombotic activity through inhibition of platelet aggregation, thereby contributing to the improvement of blood circulation.

Example  9: Antimicrobial extract, its ethyl acetate Fraction  And its 10-50% methanol solubility Fraction  Chemical properties and stability

The 10, 30, and 50% methanol soluble fractions purified from the extracts of Tungtung nodule, its ethyl acetate fraction and the ethyl acetate fraction obtained in Example 1 and Example 2 were tested for plasma stability against antithrombotic activity, Thermal stability and acid stability were confirmed. The regulated active substances showed no significant inhibition of blood clotting and platelet aggregation inhibition activity even after 1 hour heat treatment at 100 ° C, 1 hour treatment at pH 2 (0.01M HCl), 1 hour treatment in plasma, Respectively. Considering the results of the component analysis of the fraction of Example 2, the fractionation characteristics of the organic solvent, and the above stability results, the antithrombotic active substance of Tungtung Mardi is expected to be a glycoside of the phenolic compound.

Claims (5)

A pharmaceutical composition for preventing or treating thrombosis comprising an ethyl acetate fraction derived from Salicornia spp. As an active ingredient. delete delete 2. The pharmaceutical composition according to claim 1, wherein the ethyl acetate fraction is a 10-50% methanol solubility material obtained from the ethyl acetate fraction. A health functional food for preventing or ameliorating a thrombotic disease comprising an ethyl acetate fraction derived from Salicornia spp. As an active ingredient.

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