KR20150047275A - Pharmaceutical composition comprising the organic solvent fraction yielded from hot water extraction of laminaria japonica as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

In the present invention, provided are a pharmaceutical composition and healthy food product for preventing or treating thrombosis including an organic solvent fraction obtained from extracts of Laminaria japonica which is brown algae and extracted by using hot water as an active ingredient and, more specifically, a pharmaceutical composition and healthy food product for preventing or treating thrombosis including at least one fraction among hexene fraction, ethyl acetate fraction and butanol fraction as an active ingredient wherein the organic solvent fraction has good heat stability and prevents damage of blood coagulation, thereby being processed in various forms.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis, which contains an organic solvent fraction obtained from hot water extract of sea tangle, as an active ingredient, and a health functional food. TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to a method for producing brown algae ( Laminaria The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis comprising an organic solvent fraction obtained from a hot water extract of kelp japonica as an active ingredient and more specifically to a pharmaceutical composition and a health functional food, A pharmaceutical composition for the prevention or treatment of thrombosis, which contains one or more fractions of hexane fraction, ethyl acetate fraction and butanol fraction as active ingredients, 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. Therefore, blood and blood circulation are essential for the survival of human beings. In case of damage of blood vessels, blood clots should be rapidly generated to prevent blood loss. However, excessive blood clot formation in the blood vessels interferes with blood circulation, and in severe cases, blood vessels are blocked to cause serious damage to cardiovascular system and brain tissue, resulting in thrombosis. Such thrombosis is caused by the following: 1) abnormal vascular endothelial cells, 2) excessive coagulation activity of blood coagulation factors (XII factor, XI factor, IX factor, X factor etc.) and blood coagulation enzyme (thrombin, prothrombin enzyme) 4) The abnormal function of the thrombus dissolution system is known to be responsible for these causes. These causes are caused individually or in combination, resulting in excessive blood clot formation in the blood vessels. For the prevention and treatment of thrombotic diseases, currently 1) various anticoagulants such as heparin and coumarin which inhibit the activity of blood coagulation factors to inhibit thrombogenesis, 2) anti-platelet aggregation inhibitors such as aspirin Thrombocytopenia, and 3) a thrombolytic agent such as europaea, which inhibits thrombus formation by dissolving already generated thrombus. However, they are not only very expensive, but also have limited use due to hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

On the other hand, kelp (Laminaria japonica ) is a seaweed and algae of algae. It contains 91% of moisture, 1.1% of protein, 0.2% of fat, 4.2% of carbohydrate and 3.5% of ash and has a low calorie of 12 kcal / It is used as a valuable edible seafood in Korea, which has sea on three sides. In recent years, kelp-added cakes with kelp powder (Kim Eunjung et al., 2012, The Korean Journal of Food Science and Nutrition, 25: 922-929), kelp granulosa (Kwon Yulryi et al., 2012. Korean Journal of Food Preservation, 19: 525-531) (Korean Journal of Food Science and Nutrition, 24: 520-527), kelp hamburger patty (Oh Hyun Kyung et al, 2011, The Korean Journal of Food Science and Nutrition, 31: 570-579) The development of various processed foods such as the Korean Society of Industrial and Technology Technology Fall Conference, 729-732) has been actively studied

In one room, kelp is used as a medicine for preventing hypertension, arteriosclerosis, goiter, nephritis and aging. The known components of kelp include various minerals such as algin, lamiranin, alginic acid, fucoidan and iodine. The useful physiological activities include antioxidant activity (Um, Sung Hwan et al., 2010, Journal of the Korean Fisheries Society, 43: 117-124) The effect of diabetes on blood glucose and blood cholesterol (Hwang, Hye-Jin (2007), Korean Journal of Food Science and Nutrition 36: 1391-1398) , 2010, Journal of Life Science, 20: 895-810). In addition, low-molecular nitrogen compounds of blood pressure-enhancing action called laminin are known in seaweed, and alginic acid, which is a kind of polymer polysaccharide, has been reported to have functions such as lowering of serum cholesterol and release of harmful metal (Kwon, Journal of Distribution, 19: 525-531). In recent years, it has been shown that fucoidan-like sulfate-like polysaccharides promote cancer cell death (Makarenkova et al., 2012. Zhik Mikrobiol Epidemiol Immunobiol. 6: 68-75) and hyperlipemia prevention effect (Huang et al., 2010. Pharm Biol. 48: 422-426) Have been reported. On the other hand, in the case of mice fed with 5% concentration of kelp powder, the platelet aggregation did not show any significant change (Kangmin Sook et al., 2001, Korean Nutrition Society, 34: 141-149) Fucoidan, a polysaccharide sulfate, exhibits strong antithrombotic activity (Zhao et al., 2012. Thrombosis Research. 129: 771-778). To date, it has been known that the anticoagulant mechanism of Fucoidan is related to inhibition of blood coagulation enzymes (thrombin, prothrombin) and inhibition of blood coagulation factors and inhibition of platelet aggregation (Zhu et al. 2010, Thrombosis Research. 125: 419-426). In addition, other anti-thrombotic activities of other water-soluble polysaccharides bound to sulfate groups other than fucoidan have been reported (Yoon et al., 2007, Carbohydrate Research. 342: 2326-2330). However, the antithrombotic activity of a lipophilic substance which dissolves in an organic solvent rather than a complex polysaccharide type substance such as fucoidan in sea tangle has not been reported to date.

Until now, the patent related to kelp has been applied to the manufacture of sauce using kelp (Korean Patent No. 10-1310538, eel gum and its preparation method), composition for skin improvement (Korea Patent Application No. 10-2013-0076723) (Korean Patent No. 10-1249872, a method of manufacturing a pellet for accelerating bowel movement by natural materials including kelp), a beverage with a hair loss prevention effect (Korea Patent No. 10-1266729), an obesity therapeutic pharmaceutical composition (Korean Registration No. 10-1293032, a pharmaceutical composition for treatment or prevention of obesity containing kelp and sodium butyrate as an active ingredient) have been registered and disclosed. However, patents relating to thrombosis are very limited. As a patent related to thrombosis, recently, a method for producing an alginate oligosaccharide having an effect of decomposing alginic acid in sea tangle or seaweed and decomposing already formed thrombus has been patented (Korean Patent No. 10-0538553, alginate oligosaccharide A method for producing the same, an alginate oligosaccharide prepared by the method, body cosmetics containing the above-mentioned alginate oligosaccharide, and capsules for drinking), and Korea Patent No. 10-0337005 [the tea composition of the reproductive type with enhanced cholesterol and blood clotting function] The present invention discloses a tea composition which exhibits cholesterol and a thrombocyte function by adding red yeast and natto kinase to various grains, algae and mushroom powders, but does not specifically mention the anti-thrombotic effect of kelp, and Korean Patent No. 10-0345040 [Improvement agent for blood circulation containing a complex of red ginseng and method for producing the same] Discloses a blood circulation improving agent comprising a red ginseng complex obtained by extracting kelp, manure, broiler blood, licorice and the like with water and a mixture of these extracts, and a method for producing the same, wherein the improvement in blood circulation is derived from herbal medicines including red ginseng It is known that the direct antithrombotic activity of kelp is not reported. Therefore, until now, there have been no patents relating to anti-thrombotic activity of lipid-soluble substances in kelp, and especially in relation to the effect of inhibiting thrombosis, it is not known except fucoidan, a water-soluble polysaccharide.

KR 10-0538553 B KR 10-0345040 B

 Zhu et al., 2010, Higher specificity of the activity of low molecular weight fucoidan for thrombin-induced platelet aggregation. Thrombosis Research. 125: 419-426

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide an organic solvent fraction obtained by successively fractionating from a hot water extract of sea tangle used for edible and medicinal purposes, , Ethyl acetate fraction and butanol fraction as active ingredients, and a pharmaceutical composition for preventing or treating thrombotic diseases caused by inhibition of blood clotting factor and platelet aggregation inhibition, and a health functional food comprising the fraction .

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis comprising an organic solvent fraction obtained from a hot water extract of Laminaria japonica as an active ingredient.

The organic solvent fraction is preferably at least one fraction selected from the group consisting of a hexane fraction, an ethyl acetate fraction and a butanol fraction obtained by successively fractionating a hot water extract of sea tangle with hexene, ethyl acetate and butanol.

In addition, the present invention provides a health functional food containing the organic solvent fraction obtained from the hot-water extract of kelp of the present invention as an active ingredient.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the organic solvent fraction obtained from the hot water extract of kelp as an active ingredient of the health functional food can be obtained by extracting the kelp by hot water extraction, The hexane fraction, the ethyl acetate fraction and the butanol fraction thus prepared had excellent thrombin inhibitory effect and antithrombotic activity by the effect of inhibiting the blood coagulation factor, and the thrombin generation And there is an excellent effect that can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. Particularly, the organic solvent fractions obtained by successive fractionation from the hot water extract of sea tangle according to the present invention are excellent in thermal stability, and do not exhibit the direct inhibition of human thrombin or inhibition of blood coagulation factor even in an acidic condition of pH 2 and plasma, Powder, ring, tablet, etc., and can be prepared in a form that can be taken at any time. Therefore, it is an extremely useful invention in the pharmaceutical industry and the food industry.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared seaweed extract and sequential organic solvent fractions thereof for the purpose of developing a functional food material exhibiting anti-thrombotic and blood flow improving activity, and then, using human plasma, human thrombin, prothrombin and blood coagulation factors, Activity was evaluated and platelet aggregation inhibitory activity of kelp using human concentrated platelets was measured. The inhibition of thrombin coagulation enzyme, inhibition of blood clotting factor and platelet aggregation in hexane, ethyl acetate and butanol fractions, which were sequentially fractionated from hot water extract of sea tangle, The antithrombotic fraction exhibiting inhibitory effects and having a previously unknown substance characteristic was identified. These fractions are lipid soluble fractions that are free of fucoidan component, a water-soluble polysaccharide reported to exhibit antithrombotic activity. Interestingly, in the ethanol extract of kelp and its organic solvent fraction, only weak thrombin coagulation enzyme inhibition and blood coagulation factor inhibition were observed unlike hot water extract of kelp. Furthermore, the inventors of the present invention have found that the organic solvent fractions, which are the antithrombotic active ingredients recovered as described above, have excellent thermal stability and acid stability, so that the fractions can be used as a pharmaceutical composition for preventing or treating thrombosis, .

Specifically, the present inventors prepared a hot water extract of sea tangle to develop a pharmaceutical composition and a health functional food for preventing or treating thrombosis using sea tangle, and sequentially fractionated it with various organic solvents to obtain a hexane fraction, an ethyl acetate fraction , Butanol fractions and water residues were obtained and compared to thrombin time, prothrombin time and activated partial thromboplastin time (aPTT) for human plasma and human thrombin, respectively. , And the inhibition of platelet aggregation was evaluated. As a result, stronger thrombin and blood coagulation factor inhibitory activity were confirmed in hexane fraction, ethyl acetate fraction and butanol fraction of hot water extract of sea tangle than in aspirin (trade name: Protect) which is used clinically as an antithrombotic agent.

Accordingly, the present invention provides a pharmaceutical composition and a health functional food for preventing or treating thrombosis, which comprises an organic solvent fraction obtained from hot water extract of sea tangle as an active ingredient.

As a preferred example, the organic solvent fraction of the hot-water extract of sea tangle is selected from the group consisting of a hexane fraction, an ethyl acetate fraction and a butanol fraction obtained by fractionating the hot-water extract of sea tangle with hexene, then fractionating with ethyl acetate, Wherein the organic solvent fraction is at least one organic solvent fraction selected from the group consisting of:

Hereinafter, the method for producing the organic solvent fraction obtained from the hot-water extract of sea tangle of the present invention and the efficacy test will be described in more detail.

First, the inventors of the present invention prepared a hot-water extract and an ethanol extract of sea tangle, respectively, and measured thrombin time, prothrombin time, and apathy time at a concentration of 5.0 mg / ml. As a result, the ethanol extract showed no significant antithrombotic activity, but the hydrothermal extract showed strong antithrombotic activity. In particular, thrombin and blood coagulation factor inhibition showed strong antithrombotic activity.

Accordingly, the present invention provides a method for preparing a hot-water extract, comprising the steps of: preparing a hot-water extract from commercial kelp; Adjusting an organic solvent fraction such as hexane, ethyl acetate, butanol, etc., and a water residue from the hot water extract; Examining the antithrombotic efficacy and stability of the extract and fraction; And evaluating the stability of the active fraction.

As a preferred embodiment, the "kelp hot-water extract" of the present invention is obtained by extracting dried kelp with hot water at 100 ° C and filtering the extract using a filter net of 0.06 mm or less and concentrating it under reduced pressure .

As a preferred embodiment, the "organic solvent fraction" of the present invention is a fraction selected from the group consisting of hexane fraction, ethyl acetate fraction and butanol fraction obtained by successively fractionating a hot spring water extract of kelp with hexene, ethyl acetate and butanol .

The organic solvent fractions of the kelp hydrothermal extract showed excellent thrombin inhibition (prolonged thrombin time) and inhibition of blood coagulation factors (prolonged apathy time) in both the hexene fraction, ethyl acetate fraction and butanol fraction. The antithrombotic activity of the hexane fraction, the ethyl acetate fraction and the butanol fraction is 3 to 5 times the antithrombotic activity of the aspirin currently used as an antithrombotic agent, and the active fraction can replace the aspirin reported side effects such as gastrointestinal disorders .

The organic solvent fraction obtained from the hot water extract of sea tangle of the present invention may be prepared into powder by a conventional powdering process such as vacuum distillation, freeze drying, spray drying and the like. They remain active even at 100 ° C heat treatment and pH 2 in human body, and remain stable in plasma.

The organic solvent fraction obtained from the hot-water extract of sea tangle of the present invention can be used for preventing or treating various diseases related to 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 comprising the organic solvent fraction obtained from the hot water extract of sea tangle according to the present invention may be administered orally or rectally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, The composition can be formulated into various forms such as formulations, injections of sterilized injection solutions, and the like, and can 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, Mineral oils 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.

The pharmaceutical composition 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 organic solvent fraction obtained from the hot water extract of sea tangle as an active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex and body weight of the patient, and is generally 1 to 5,000 mg per kg of body weight , Preferably 100 to 3,000 mg per day or every other day, or one to three divided doses per 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 organic solvent fraction obtained from the hot water extract of tangle of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they may be powder, granules, It can be used in beverage form.

The organic solvent fraction obtained from the hot water extract of sea tangle according to the present invention may be added in an amount of 0.01 to 15% by weight of the whole food, and the health beverage composition may be added in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, Can be added.

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 flavor agent include tau martin, rebaudioside A, glycyrrhizin, saccharin, and aspartame. The proportion of the above-mentioned flavoring agent 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 organic solvent fraction obtained from the hot-water extract of sea tangle of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. The following examples are only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[ Example ]

Example  1: Kelp Heat number  Extracts and their organic solvents Fraction  pharmacy

In 2013, hot water and ethanol extracts were prepared from Gyeongnam dried sea tangle, supplied from Andong Kanggak language co., Ltd. The ethanol extracts were extracted three times at room temperature for 8 hours with 10 times volume of ethanol (95%, Duksan, Korea) to dry kelp. The hot - water extracts were prepared by adding 10 times volume of water to dried kelp and subjected to high temperature extraction three times at 100 ° C for 1 hour. Each of the extracts was recovered after removing the foreign substances by using a filter machine, and concentrated under reduced pressure to prepare powder, which was stored at low temperature until use. Thereafter, the ethanol extract and the hot water extract were suspended in distilled water, and then fractionated with hexane, ethyl acetate, and butanol sequentially to obtain a final water residue.

The yields of ethanol extract and hot water extract of sea tangle were 9.1 ± 0.7% and 50.5 ± 1.2%, respectively, and the hot water extract was 5 times more than the ethanol extract. The yields of hexane fraction, ethyl acetate fraction, butanol fraction and water residue of hot water and ethanol extract of sea tangle were as shown in Table 1. In the case of hot water extract, 96.5% was water residue and 3.5% of organic solvent fraction , And ethanol extracts showed 84.8% of water residue and 15.2% of organic solvent fraction.

Kelp Heat number  And ethanol extracts and their organic solvent fractionation efficiencies division Extraction Efficiency or Fraction Efficiency (%) Extraction solvent extract Hexene fraction Ethyl acetate
Fraction Butanol fraction Water residue Kelp Heat number 50.5 ± 1.2 0.1 0.8 2.6 96.5 ethanol 9.1 ± 0.7 5.5 2.0 7.7 84.8

Example  2: Seaweed extract and organic solvent Fraction  Component analysis

Total flavonoid, total polyphenol, total sugar, and reducing sugar contents were measured in hot water and ethanol extracts and their fractions. To determine the total flavonoid content, each sample was stirred for 18 hours in methanol, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extract, 40 μl of 1N NaOH was added, and the reaction was conducted at 37 ° C. for 1 hour and then absorbance was measured at 420 nm . As a standard reagent, rutin was used. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ The saturated solution was added and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was quantified by phenol-sulfuric acid method. The results are shown in Table 2 below.

Sea tangle extract and organic solvent Fraction  Component analysis division sample Content (mg / g) Total flavonoid Total polyphenol Total Reducing sugar Kelp / hot water Hot water extract 0.50 + - 0.73 1.32 + 0.02 40.32 ± 0.59 14.78 ± 2.04 Hexene fraction 0.05 ± 0.08 1.15 ± 0.02 14.81 ± 0.48 8.11 ± 0.90 Ethyl acetate
Fraction 0.32 ± 0.01 4.15 + 0.04 14.06 + - 0.44 9.60 ± 0.00 Butanol fraction 0.02 ± 0.01 2.70 + - 0.01 16.24 ± 0.28 11.13 ± 0.24 Water residue 0.01 ± 0.04 1.56 + 0.06 42.95 ± 0.40 18.90 ± 0.78 Kelp / ethanol Ethanol extract 0.02 0.03 0.30 0.01 9.99 ± 0.79 8.87 ± 0.06 Hexene fraction 0.75 + - 0.68 2.18 ± 0.14 45.37 + - 2.46 31.77 ± 0.96 Ethyl acetate
Fraction 0.29 ± 1.33 4.39 + 0.02 84.92 + - 2.26 35.59 ± 0.36 Butanol fraction 0.02 0.03 1.06 ± 0.05 13.38 + - 0.12 9.13 + 0.06 Water residue 0.03 ± 0.01 0.03 ± 0.01 9.01. + -. 0.32 6.71 ± 0.24

As shown in Table 2, the total amount of total flavonoids and total phenol components were higher in the case of the hot-water extract of kelp, especially the ethanol extract, and most of the sugar in the hot-water extract was converted into water residue. However, Hexene and ethyl acetate fractions, indicating that the organic solvent fractions of hydrothermal and ethanol extracts were significantly different.

Example  3: Seaweed extract and organic solvent Fraction  Evaluation of blood coagulation inhibitory activity

The blood coagulation inhibitory activity of kelp extract and fractions was evaluated. The blood coagulation inhibitory activity was evaluated according to the previously reported method (Li et al. 2010. J. Life Science , 20. 922-928), thrombin time, prothrombin time and apathy time were measured. The specific measurement method is as follows.

Thrombin  time( Thrombin Time )

50 [mu] l 0.5 U thrombin (Sigma Co., USA) and 20 mM CaCl ₂ (Control plasma, Thermo Fisher Scientific Inc, MA, USA) was added to the tube, and then the plasma was incubated for 1 hour until the plasma coagulated The time 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 results were expressed as the mean value of three repeated experiments, and the coagulation inhibition activity was expressed as the thrombin time at the time of adding the sample divided by the thrombin time at the solvent control.

Prothrombin  time( prothrombin time )

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample were added to a tube of Amelung coagulometer KC-1A (Japan) and incubated at 37 ° C. for 3 minutes. Then, 130 μl of PT reagent was added, Scientific Inc, MA, USA) was used as the average value of the experiment in which the time until solidification was 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 results were expressed as the mean value of three repeated experiments, and the coagulation inhibition activity was expressed as the prothrombin time at the time of adding the sample divided by the prothrombin time at the solvent control.

aPTT  ( activated Partial Thromboplastin Time )

Plasma (control plasma, Thermo Fisher Scientific Inc , MA, USA) 100μl with the sample 10μl Amelung coagulometer KC-1A (Japan ) was added to the tube and then heated at 37 ℃ 3 bungan, 50μl of the aPTT reagent (Sigma, ALEXIN ^TM of , USA) and incubated at 37 ° C for 3 minutes. After the addition of 50 μl CaCl ₂ (35 mM), 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 were expressed as the mean value of the experiment which was repeated three times, and the coagulation inhibition activity was expressed as the apitime time when the sample was added divided by the apitime time of the solvent control.

The blood coagulation inhibitory activity of the kelp hydrothermal extract and fractions is shown in Table 3. On the other hand, seaweed ethanol extract and its fractions did not show significant blood clotting inhibitory activity (no result).

Kelp Heat number  Extracts and organic solvents Fraction  Blood coagulation inhibitory activity sample Concentration (mg / ml) Blood coagulation inhibitory activity ^* Thrombin time Prothrombin time Apathy time DMSO - 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 aspirin 1.5 1.8 ± 0.0 1.6 ± 0.1 1.7 ± 0.0
Heat number
extract 5.0 > 15.0 > 15.0 > 15.0 2.5 > 15.0 4.7 ± 0.1 > 15.0 1.2 > 15.0 3.1 ± 0.1 > 15.0 0.6 > 15.0 1.6 ± 0.1 > 15.0
Hexen
Fraction 5.0 > 15.0 2.1 ± 0.2 12.4 ± 0.5 2.5 8.7 ± 0.1 1.3 ± 0.0 5.9 ± 0.1 1.2 5.1 ± 0.1 1.0 ± 0.2 2.9 ± 0.2 0.6 3.3 ± 0.0 1.0 ± 0.1 2.3 ± 0.1
Ethyl acetate
Fraction 5.0 > 15.0 3.2 ± 0.1 > 15.0 2.5 > 15.0 2.1 ± 0.1 7.7 ± 0.4 1.2 8.0 ± 0.2 1.4 ± 0.1 3.4 ± 0.2 0.6 4.3 ± 0.1 1.1 ± 0.2 2.5 ± 0.0
Butanol
Fraction 5.0 9.6 ± 0.2 1.4 ± 0.1 3.6 ± 0.2 2.5 4.1 ± 0.0 1.0 ± 0.1 2.8 ± 0.1 1.2 2.7 ± 0.1 1.0 ± 0.1 1.8 ± 0.1 0.6 2.0 ± 0.1 1.0 ± 0.1 1.3 ± 0.2
water
Residue 5.0 > 15.0 > 15.0 > 15.0 2.5 > 15.0 4.3 ± 0.5 > 15.0 1.2 > 15.0 3.3 ± 0.1 > 15.0 0.6 > 15.0 1.8 ± 0.0 > 15.0

Blood coagulation inhibition activity ^* : Coagulation time at sample addition divided by coagulation time of solvent control

As shown in Table 3, aspirin used as an antithrombotic agent prolonged thrombin time, prothrombin time, and apathy time by 1.8, 1.6, and 1.7 times, respectively, at a concentration of 1.5 mg / ml.

On the other hand, in the hot water extract of sea tangle, the thrombin time and apathy time were prolonged more than 15 times as compared with the solvent control at the concentration of 0.6 mg / ml, and the antithrombotic activity was almost the same even after the organic solvent fraction.

However, the hexane, ethyl acetate and butanol fractions, which are organic solvent fractions, exhibited a remarkable antithrombotic activity, which was not known until now. Particularly in the ethyl acetate fraction, thrombin time, prothrombin time, Fold, 2.1 times, and 7.7 times, respectively, and 8.0 times, 1.4 times, and 3.4 times, respectively, at the concentration of 1.2 mg / ml to confirm the best blood coagulation inhibitory activity among the fractions. Ethyl acetate fraction of kelp hydrothermal extract was only 0.4% of dry seaweed, but it could be used as an antithrombotic agent with excellent novel activity.

On the other hand, no blood coagulation inhibitory activity was found in the ethanol extract of the sea tangle and its fractions. Especially, in the ethyl acetate fraction of the ethanol extract, the antithrombotic activity hardly appeared even at the concentration of 5 mg / ml. These results indicate that the antithrombotic active fraction of kelp shows excellent antithrombotic activity only in the solvent extraction using hot water and its specific organic solvent fraction. In addition, the antithrombotic activity by water-soluble polysaccharides of kelp is well known, but lipophilic substances, especially hexene fraction, ethyl acetate fraction and butanol fraction, are not known. Therefore, You can expect it.

Example  4: Seaweed extract and organic solvent Fraction  Human platelet aggregation inhibitory activity

To evaluate the antithrombotic activity of kelp extract and fractions, aggregation inhibitory activity against human platelets was evaluated, and the evaluation method was as follows.

Platelet Aggregation Inhibitory Activity Platelet aggregation inhibition activity )

Platelets were mixed with 3.2% sodium citrate solution in a ratio of 1: 9 from healthy human whole blood, centrifuged at 1,100 rpm for 10 minutes, and the upper layer of platelet rich plasma (PRP) to _{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) and washed once. After resuspending in 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 , 025% gelatin, pH 7.4) After centrifugation for several minutes, the cells were resuspended in a suspending buffer, and the platelet count was adjusted to ⁴ × ^{10 9} / ml. Platelet aggregation was measured at 37 ° C using a whole-blood agarometer (Chrono-log, USA). The platelet aggregation was measured by cohesive strength, slope, elongation time, and falling area .

The results of measuring the inhibition of platelet aggregation of hot water extract and organic solvent fraction of sea tangle are shown in Table 4 below.

Kelp Heat number  Extracts and organic solvents Fraction  Human platelet aggregation inhibitory activity division sample
(0.25 mg / ml) Platelet Aggregation Inhibitory Activity ^* burglar
(Amplitude:
ohm) inclination
(Slope) Extension time
(Lag time: seconds) Fall area
(Area
under) Relative Flocculation Rate (%) Kelp Hot water extract 28 6 18 226.6 183.4 Hexene fraction 13 2 33 97.0 78.5 Ethyl acetate fraction 15 2 17 122.1 98.9 Butanol fraction 14 2 31 105.7 85.6 Water residue 26 6 17 211.6 171.3 DMSO (DMSO) 18 3 16 124.3 100.0 Aspirin (0.25 mg / ml) 8 One 39 49.8 40.4 Aspirin (0.5 mg / ml) 4 0 52 30.1 24.4

Platelet aggregation inhibition activity ^* : The smaller the cohesion strength, the slope and the lowering area, the better the coagulation inhibition. The longer the extension time, the better the coagulation inhibition

As shown in Table 4, aspirin exhibited excellent human platelet aggregation inhibitory activity and ascertained concentration dependent coagulation inhibitory activity. However, the hot water extracts of kelp and the water residues after organic solvent fraction were found to promote platelet aggregation rather. This suggests that fucoidan, a conventional water-soluble polysaccharide, promotes platelet aggregation (Zhu et al., 2010, Thrombosis Research. 125: 419-426). On the other hand, inhibition of platelet aggregation in the hydrothermal extract fractions was in the order of hexene fraction, butanol fraction, and ethyl acetate fraction, but showed weak activity as a whole.

Analysis of the results of Example 3 and Example 4 showed that even though the hot water extract of sea tangle and the water residue (accounting for 96.5% of the total) after the organic solvent fraction inhibited thrombus formation through strong anticoagulant activity, It is difficult to expect a real antithrombotic effect because of its strong platelet aggregation promoting activity. This is consistent with a report in which no significant change in blood flow improvement was observed in mice fed a 5% concentration of kelp powder (Kang, Min-Sook et al., 2001, The Korean Nutrition Society, 34: 141-149). However, the hexane fraction, ethyl acetate fraction and butanol fraction obtained sequentially from the kelp hydrothermal extract do not show platelet aggregation promoting activity while exhibiting excellent blood coagulation inhibition, so that actual antithrombotic activity is expected. In particular, the ethyl acetate fraction of the kelp hydrothermal extract was high in yield and strongest in activity, and it was confirmed that the antithrombotic agent was expected to be developed as a novel antithrombotic agent in the future, and that the activity was due to inhibition of thrombin and blood coagulation factors.

Example  5: Kelp Heat number  Sequence from extract Fractionated Hexen , Ethyl acetate and butanol Fraction  Chemical properties and stability of active materials

The hematopoietic activity of the recovered active substance was confirmed after the crude hot water extract and the hexane fraction, the ethyl acetate fraction, the butanol fraction and the water residue of the tuna hot-water extract obtained in Example 1 were subjected to a pressure-drying to prepare a powder, The fractions were further tested for plasma stability, thermal stability and acid stability.

The active substances of the adjusted hexene fraction, ethyl acetate fraction and butanol fraction showed no hemolytic activity on human erythrocytes up to a concentration of 0.5 mg / ml (Table 5), and in particular, 2 hours treatment, 2 hours treatment in pH 2 (0.01 M HCl) and 2 hours treatment in plasma showed no significant decrease in blood coagulation inhibition activity measured by apitime (Table 6) .

Kelp Heat number  Extracts and organic solvents Fraction  Human red blood cells Hemolytic activity The sample (0.5 mg / ml) Human hemolytic hemolytic activity (%) Hot water extract 0.6 ± 0.7 Hexene fraction 3.3 ± 0.2 Ethyl acetate fraction 3.1 ± 0.4 Butanol fraction 3.2 ± 0.8 Water residue 3.2 ± 0.8 Triton X-100 (0.1% 100 ± 0.0 Amphotericin B 0.1 mg / ml 100 ± 0.0

Kelp Heat number  The ethyl acetate extract Fraction  stability Treatment (1.2 mg / ml) Blood coagulation inhibitory activity ^* APT (before processing) APT (after processing) 100 캜, 2 hours 7.7 ± 0.4 ^a 7.5 ± 0.5 ^a pH 2. 2 hours 7.7 ± 0.4 ^a 7.2 ± 0.5 ^a Plasma, 2 hours 7.7 ± 0.4 ^a 7.7 ± 0.3 ^a

Blood coagulation inhibition activity ^* : The value obtained by dividing the apical time at the time of adding the sample by the apical time of the solvent control

Claims (3)

Sea tangle ( Laminaria japonica) a pharmaceutical for the prevention and treatment of thrombosis comprising the organic solvent fraction obtained from the hot-water extract as an active ingredient compositions. [Claim 2] The method according to claim 1, wherein the organic solvent fraction is a fraction selected from the group consisting of a hexane fraction, an ethyl acetate fraction and a butanol fraction obtained by successively fractionating hot water extract of sea tangle with hexene, ethyl acetate and butanol, Or a pharmaceutically acceptable salt thereof. A health functional food comprising the organic solvent fraction according to any one of claims 1 to 6.