KR101622259B1 - Anti-thrombotic composition comprising phenol compound - Google Patents

Abstract

The present invention relates to an antioxidant composition comprising a phenol compound as an active ingredient. The phenol compound according to the present invention is excellent in anticoagulant activity for inhibiting the coagulation of plasma induced by thrombin action and is more useful as an antithrombotic agent than antipyretic agent such as aspirin.

Description

An anti-thrombotic composition comprising phenol compound as an active ingredient,

The present invention relates to an antioxidant composition comprising a phenol compound as an active ingredient.

Recently, due to the spread of well-being culture, the size of health functional food market has been increasing. In Korea, it has been rapidly increasing year by year to 1.9 trillion won in 2008, 1.202 trillion won in 2009, and 1.32 trillion won in 2010, So many consumers are looking for more than 2 trillion won. In addition, the overseas market is rapidly growing and growing every year due to the continuous development of science and technology, the global population aging, the global economic growth and the universal interest in human health.

Cardiovascular disease is a disease that involves heart-related diseases (myocardial infarction, hypertension, heart failure, arrhythmia, arteriosclerosis) and vascular disease (stroke, peripheral vascular disease). According to statistics of the World Health Organization (WHO) Despite the remarkable progress, it has been reported that the incidence rate and prevalence rate have increased steadily over the last 30 years. It is a kind of fatal disease that accounts for a very high rate of mortality due to cardiovascular system diseases in Korea and worldwide .

The increase in the incidence and mortality rate of cardiovascular diseases is mainly due to the westernization of economic growth and lifestyle, as well as changes in dietary habits such as high fat diet, increased stress, aging population due to extended life span, and lack of exercise. It is pointed out that serious diseases such as cardiovascular diseases are caused by the diseases themselves and serious physical and social sequelae after the disease, resulting in overpayment of the national medical expenses and deterioration of the quality of life. Therefore, as the problems related to such cardiovascular diseases become increasingly recognized, there is a great investment in researches on preventive and therapeutic treatment in addition to studies on the pathogenesis mechanism of these diseases worldwide. As a result, new blood vessels As a part of research for the development of thrombotic agents, researches have been actively carried out to find new materials exhibiting anticoagulant activity or fibrinolytic activity.

However, in spite of these researches and developments related to cardiovascular diseases, the prevalence rate and incidence of cardiovascular diseases have been increasing steadily throughout the world. Now, the development of therapeutic drugs for related diseases and the prevention and prevention of cardiovascular diseases There is an urgent need for the development of active materials and food materials that can be used.

On the other hand, thrombus is an aggregation of blood factors, mainly consisting of aggregates of platelets and fibrin, and is usually formed to prevent excessive bleeding of blood vessels. When the platelets come into contact with the expression of the subcutaneous layer, a thrombus is formed to initiate a reaction to block the vasculature. This process is called hemostasis, and this process is very important to prevent excessive bleeding from the wound site. Arterial thrombosis causes serious disease due to local occlusion, whereas venous thrombosis mainly causes distant occlusion, or embolization. Therefore, the medicament which inhibits platelet aggregation inhibition or thrombolytic action can be used for the treatment of diseases associated with thrombus and embolism, such as myocardial infarction, angina pectoris, thrombotic phlebitis, arterial embolism, coronary and cerebral arterial thrombosis, peripheral vascular or deep vein thrombosis, Pathologic symptoms after thromboembolism and subarachnoid haemorrhage, percutaneous coronary angioplasty (PTCA), coronary artery bypass grafting (PTCA), coronary artery bypass grafting ) And stent insertion, stent restenosis after stent placement, catheter thrombotic occlusion or reocclusion, acute coronary syndrome, TIA (transient ischemic attack or acute cerebral angiopathy syndrome), heart failure, chest due to ischemic pathology Pain syndrome, X syndrome, diabetes mellitus. There are blood coagulation and thrombus formation mechanisms in the blood vessel blocking mechanism.

The blood coagulation mechanism changes the soluble fibrinogen to an insoluble fibrin and the blood coagulates. The main initiator is the exogenous factor tissue factor (TF) and the exogenous factor VIIa factor (factor VIIa) (Pia Siljander 1-80, HELSINKI 2000). At each stage, several clotting factors are activated to form a fibrin colt (cross-linked fibrin molecule), which in turn produces thrombin, which stimulates the release of the activating factor by a strong platelet active.

The thrombus formation mechanism is a voluntary process to prevent hemorrhage from damaged blood vessels, not to normal vascular endothelial cells, since platelets adhere to collagen upon impaired vascular endothelium formation and form a viscous gelatin mass by mutual activation.

Drugs such as thrombolytic agents and antiplatelet agents are known to remove these blood clots. The thrombolytic agent is a drug that rapidly cleaves thrombus by catalyzing the formation of serine protease plasmin from plasminogen. It is a drug that can be used to treat streptokinase, urokinase, APSAC, t- PA, and the antiplatelet agents regulated by Group 3 substances are catecholamine, collagen, thrombin, prostacyclin, which is produced outside the platelets and acts on platelet membrane receptors; ADP, PGD2, PGE2, serotonin, which are produced in platelets and act on membrane receptors; And it is generated in the platelet substances that act on the inner platelet prostaglandin endo peroxide (prostaglandin endoperoxide), thromboxane A2 (TXA2), cAMP, cGMP has, Ca ^{+ 2.} The antiplatelet agent is also known as a calcium ionophore, which binds to the cell membrane to form calcium channel into the cell through calcium to induce aggregation. A 23187 and a thromboxane A2 analog (thromboxane A2 analog) (Tina Ritz, et al., International Journal of Biochemistry & Cell Biology, 36: 1187-1205, 2004), Norimichi Nakahata, Satoko Ohkubo, Emiko Ito, Toxicon, 37: 1375-1389, 1999). However, the thrombolytic agents or antiplatelet agents developed so far have problems causing various adverse effects such as hemostatic hypertrophy, infertility, and digestive disorders.

On the other hand, as a part of prevention and treatment of cardiovascular diseases, the 21st century type health functional food which is responsible for human health in the future has been greatly interested in the world and many studies have been conducted. Grains are the most familiar food that has been around for the longest time.

Among these cereals, cereals have recently been reported to contain physiological activities such as antioxidation, anticancer, anti-inflammation, and anti-diabetic activity. Thus, the possibility of using them as health functional foods is expected to be high in the future, It is true. Especially, in relation to the health functional properties of sorghum, the inhibitory activity of the extracts of Sui extract (Choi et al., 1998), antifungal and antipyretic activity (Lee et al., 2000), spleen cell and macrophage activity enhancing activity (Kim et al., 2006), antioxidant and antimutagenic activity (Kwak et al., 2004), antioxidant and antimicrobial activity (Kil et al., 2009) et al., 2011).

However, although studies on the health function of the grains used as food have recently been carried out variously, studies on the antithrombotic effect of sorghum, especially the effects of phenolic compounds, which are generally known as antioxidative effects, have not yet been made There is no research on this.

Accordingly, the inventors of the present invention confirmed that the gold extract of ethanol extract of the present invention has an excellent anticoagulant activity for inhibiting the coagulation of plasma induced by thrombin action during the research for antithrombogenic effect search and development of antithrombogenic material, Derived phenol compound exhibited better antithrombotic activity than aspirin, thereby completing the present invention.

It is an object of the present invention to provide a composition for exclusive use in blood circulation which contains a phenol compound as an active ingredient.

And a composition for preventing or treating a thrombotic disease comprising the phenolic compound of the present invention as an active ingredient.

Accordingly, the present invention provides a composition for exclusive use of an antihypertensive agent comprising a phenol compound as an active ingredient.

The present invention also provides a composition for the prevention or treatment of thrombotic diseases comprising a phenol compound as an active ingredient

The phenolic compound of the present invention is excellent in anticoagulant activity for inhibiting the coagulation of plasma induced by thrombin action and is more useful as an antithrombotic agent than antipyretic agent such as aspirin.

FIG. 1 is a view showing a process of extracting a golden bug extract. FIG.
FIG. 2 is a graph showing the thrombin inhibitory activity of a phenol compound derived from a golden raspberry ethanol extract. FIG.
FIG. 3 is a graph showing thrombin inhibitory activity according to the concentration of a phenol compound isolated from an extract of golden rumen ethanol according to the present invention.
FIGS. 4 and 5 are graphs showing the thrombopathlcin inhibitory activity of the phenol compounds isolated from the golden waxy ethanol extract according to the present invention. FIG.
6 and 7 are graphs showing the prothrombin inhibitory activity of the phenol compounds isolated from the golden wool ethanol extract according to the present invention.

The present invention relates to the use of m-Coumaric acid, Formononetin, Homogentisic acid and Pyrogallol, [beta] -resorcylic acid, Salicinic acid Wherein the composition contains at least one phenolic compound selected from the group consisting of salicylic acid, benzoic acid, and t-cinnamic acid as an effective ingredient.

The present invention also relates to a process for the preparation of a pharmaceutical composition comprising m-Coumaric acid, Formononetin, Homogentisic acid and Pyrogallol,? -Resorcylic acid, There is provided a composition for preventing or treating thrombotic diseases comprising, as an active ingredient, at least one phenol compound selected from the group consisting of salicylic acid, benzoic acid and t-cinnamic acid do.

The composition comprises a pharmaceutical composition or a food composition.

Hereinafter, the present invention will be described in more detail.

The phenolic compounds according to the present invention are represented by the following formulas (1) to (8).

[Formula 1] m- Kumar  The acid (m- Coumaric acid )

[Chemical Formula 2] Formononetin ( Formononetin )

(3) Homogenethicine  mountain( Homogentisic acid )

[Chemical Formula 4] Pyrogallol ( Pyrogallol )

[Formula 5]? - Resozil  The acid (? Resorcylic acid )

[Chemical Formula 6] Salicinic acid ( Salicylic acid )

(7) Benzo  mountain( Benzoic acid )

[Chemical Formula 8] t-cinnamic acid (t- Cinnamic acid )

The phenol compounds of the formulas (1) to (8) of the present invention can be purchased commercially or synthesized by an organic synthesis method or can be used separately from natural products. In the present invention, it was extracted and separated from the golden wastes.

More specifically, after gold polishes have been _ground , water, C ₁ -C ₄ Alcohol or a mixed solvent thereof to obtain a golden barley crude extract. The C ₁ -C ₄ The alcohol is preferably 1 to 100% methanol or 1 to 100% ethanol, more preferably 1 to 100% ethanol. The golden waxy crude extract is centrifuged to recover the supernatant, and the supernatant is concentrated under reduced pressure and dried to obtain a golden waxy extract in powder form. Then, the gold-tracing extract is sequentially extracted with n-hexane, methylene chloride, ethyl acetate, and n-butanol, and this is concentrated under reduced pressure to obtain each organic solvent fraction. The fractions were then subjected to HPLC analysis to isolate the phenolic compounds of Formulas 1-8 of the present invention.

It was confirmed that the phenol compounds of the formulas (1) to (8) according to the present invention had better thrombin inhibitory activity than the other phenol compounds.

The phenol compound according to the present invention has an excellent effect of inhibiting thrombin-induced thrombus formation, and thus can be effectively used as a composition for antihypertensive and a composition for preventing or treating thrombotic diseases.

The thrombotic disease is selected from the group consisting of myocardial infarction, angina pectoris, thrombotic phlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, peripheral vascular thrombosis, deep vein thrombosis, arterial thrombosis, venous thrombosis, ischemic cerebral infarction, arteriosclerosis, hypertension, pulmonary hypertension, (PTCA) or stent implantation, restenosis after stent placement, catheter thrombosis, coronary artery occlusion, and / or atherosclerosis. Including, but not limited to, one or more selected from the group consisting of acute coronary syndrome, TIA (transient ischemic attack or acute cerebral vascular syndrome) and heart failure.

The composition of the present invention may contain at least one known active ingredient having a prophylactic or therapeutic effect of antithrombotic or thrombotic diseases together with a phenol compound.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. 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. Examples of suppository bases include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, and can be appropriately selected by those skilled in the art. For the desired effect, the phenolic compound of the present invention can be administered in an amount of 1 μg / kg to 1000 mg / kg per day. The composition may be administered once a day, or divided into several doses.

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

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of anti-thrombotic or thrombotic diseases.

In the present invention, a food composition refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease, and aging inhibition.

The phenolic compound of the present invention may be added to a health functional food for the purpose of preventing or improving antithrombotic or thrombotic diseases. When the phenol compound of the present invention is used as a food additive, the phenol compound may be added as it is, or may be used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the phenolic compound derived from the golden wax of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred embodiments and production examples are shown to facilitate understanding of the present invention. However, the following Examples and Preparation Examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the Examples and Production Examples.

[ Example  1] Separation of Phenolic Compounds from Gold Extracts

1. Preparation of Golden Chrysanthemum Ethanol Extract and Fraction

One kilogram of seeds and blood - filled golden pellets were supplied from the National Institute of Food and Nutritive Research, Korea (Milyang, Kyungnam). In order to identify the active ingredient contained in the millet, 500 g of the golden waxy dressing was ground with Blender 7012 (Dynamics corporation, USA) for 10 minutes, and then mixed with 80% ethanol. Thereafter, extraction (three times with 1000 ml, three times at a time) was carried out using an 80 ° C reflux condenser. The ethanol extract was centrifuged at a speed of 10,000 rpm for 20 minutes. The supernatant was recovered, concentrated under reduced pressure using a rotary vacuum evaporator, and dried to obtain a golden waxy ethanol extract in powder form. After that, the golden extract of ethanol was dissolved in 500 ml of water, extracted three times each with the same amount of n-nucleic acid, methine chloride, ethyl acetate and n-butanol, respectively, and concentrated under reduced pressure to obtain each organic solvent fraction . Each step-by-step extraction process is shown in FIG. 1, and the results are shown in Table 1.

Golden Chrysanthemum Extract and Fraction Weight (g) One 80% ethanol 7.7 2 n-hexane 3.35 3 Methylene chloride 0.36 4 Ethyl acetate 0.85 5 n-butanol 1.50 6 The water layer 2.48

2. A gold rally  Ethanol extract HPLC  analysis

Fractions of the ethanol extract were subjected to HPLC analysis. Specifically, the peak area of the phenol compound contained in the 80% ethanol extract of the golden pickles was compared with the peak area of the standard material, and the content of each constituent was determined.

The results of HPLC analysis of the 80% ethanol extract of the golden wax are shown in Table 2.

As shown in Table 2, it was confirmed that various kinds of phenol compounds, including the ethanol extract of golden rices, were contained.

No. compound μg / mg  content EtOH Hexane M.C. EtOAc BuOH H ₂ O One Pyrogallol 0.04 0.03 0.05 2 Gallic acid 0.17 0.08 0.13 3 Homogentisic acid 0.08 0.20 4 4-Hydroxybenzoic acid (4-hydroxybenzoic acid) 0.32 0.06 0.06 2.62 0.07 5 (+) - Catechin (Catechin) 1.90 0.15 0.15 5.24 0.50 6 Chlorogenic acid 0.36 0.07 7 Caffeic acid 0.49 0.05 0.08 1.42 0.29 8 Syringic acid 0.10 9 Isoorientin 1.81 0.26 0.25 3.45 0.49 10 Orientin 0.72 0.77 0.74 1.39 11 p - Coumaric acid 0.25 0.07 0.06 0.68 0.82 0.14 12 Ferulic acid 0.08 0.10 0.86 0.87 13 Veratric acid 1.10 0.36 0.33 4.83 0.22 0.46 14 Hesperidin 1.10 0.46 0.42 3.91 0.93 15 o - Coumaric acid 0.17 0.09 0.08 1.35 0.61 16 Protocatechuic acid 1.31 1.00 0.94 17 Resveratrol 1.40 0.51 0.69 0.44 18 Salicylic acid 1.76 1.90 1.83 1.73 0.21 19 t - sinnan acid (t -Cinnamic acid) 0.45 0.08 0.09 0.14 0.06 0.03 20 Apigenin 0.89 4.03 3.96 3.33 0.14 0.52 21 Hesperitin 0.33 0.22 2.90 0.31 0.80 22 σ-Vinylguacol (σ-Vinylguacol) 0.60 0.13 0.13 6.72 23 Formononetin 0.94 0.25 0.18 0.01 24 Biochanin A 0.15 0.14 25 Gentisic acid 2.13 8.73 1.16 26 Apigenidin 0.27 3.61 1.15 27 Naringin 1.33 1.84 1.33 28 Myricetin 0.23 7.00 0.38 29 Quercetin 9.29 4.66 1.11 7.27 2.13 30 Naringenin 2.60 0.49 4.50 1.59 2.09 31 Kaempferol 2.90 0.43 2.53 1.17 6.88 32 Vanillic acid 1.20 33 Rutin 0.12 34 Benzoic acid 0.15 35 beta -resorcylic acid < / RTI > 0.19 36 Kumar acid m- (m -Coumaric acid) 0.08

[ Experimental Example  1] The phenol compound of the present invention Anti-thrombosis  Effect measurement

One. From the Golden Chapters  Of the isolated phenol compound Thrombin  Measurement of inhibitory activity

In the case of TT (Thrombin time) measurement, plasma coagulation time (thrombin time) was measured using an automatic blood coagulation analyzer (Sysmex CA-540, Japan) according to the previously reported method of Thompson and Harker Respectively.

More specifically, 10 占 퐇 of the single phenolic compound storage solution prepared in Example 1 dissolved in DMSO and 50 占 퐇 of human thrombin (10 units / ml PBS) were preliminarily mixed and preincubated at 37 占 폚 for 10 minutes . Then, 50 μl of 20 mM CaCl ₂ solution and 100 μl of plasma were added and the time elapsed until the plasma was solidified, that is, the plasma coagulation time (thrombin time), was stored at 37 ° C. Aspirin (Sigma Chemical Co.) was dissolved in DMSO at concentrations of 7.5 mg / ml and 10 mg / ml, and the anti-thrombin inhibitory activity-based anti-DMSO was used as a negative control. The thrombocyte activity was calculated by the following equation (expressed as an average value of plasma coagulation time measured repeatedly at least three times).

[ Equation  One]

Thrombin inhibitory activity (%) = (coagulation time in sample treatment / coagulation time in negative control) x 100

The thrombin inhibitory activity of the phenol compounds of the present invention is shown in FIG. 2 and Table 3, and the concentration-dependent thrombin inhibitory activity of the phenol compounds having excellent thrombin inhibitory activity in the above Table 3 is shown in Table 4.

compound Low performance (%) One DMSO 100 2 Aspirin 360 / / ml 124 3 Aspirin 480 [mu] g / ml 167 4 Tocopherol 480 [mu] g / ml 116 5 Benzoic acid 480 [mu] g / ml 185 6 Biochanin A 480 [mu] g / ml ∞ 7 Caffeic acid 480 / / ml 88 8 480 / / ml of chlorogenic acid 105 9 t-Cinnamic acid 480 [mu] g / ml 176 10 m-Coumaric acid 480 [mu] g / ml 190 11 o-Coumaric acid 480 [mu] g / ml 94 12 480 [mu] g / ml of p-Coumaric acid 167 13 Ferulic acid 480 [mu] g / ml 159 14 Formononetin 480 [mu] g / ml 170 15 Gallic acid 480 [mu] g / ml 96 16 Gentisic acid 480 [mu] g / ml 100 17 Homogentisic acid 480 [mu] g / ml 188 18 480 [mu] g / ml of Hesperidin 96 19 Hesperetin 480 [mu] g / ml 95 20 p-Hydroxybenzoic acid 480 占 퐂 / ml 124 21 480 [mu] g / ml Kaempferol ∞ 22 Myricetin 480 [mu] g / ml 180 23 Naringenin 480 [mu] g / ml 92 24 Naringin 480 [mu] g / ml 176 25 Protocatechuic acid 480 [mu] g / ml 95 26 Pyrogallol 480 [mu] g / ml 188 27 β-Resorcylic acid 480 μg / ml ∞ 28 Resveratrol 480 [mu] g / ml 98 29 Rutin 480 [mu] g / ml 104 30 Quercetin 480 [mu] g / ml 94 31 Salicylic acid 480 / / ml ∞ 32 Sinapic acid 480 [mu] g / ml 92 33 Syringic acid 480 [mu] g / ml 86 34 Vanillic acid 480 [mu] g / ml 105 35 Vanillin 480 [mu] g / ml 107 36 Veratric acid 480 [mu] g / ml 107

compound Low performance (%) One DMSO 100 2 Aspirin 360 [mu] g / ml 112 3 Aspirin 480 [mu] g / ml 157 4 Benzoic acid 360 / / ml 159 5 Benzoic acid 480 [mu] g / ml 185 6 Biocanin A 360 ㎍ / ml 173 7 Biochanin A 480 / / ml ∞ 8 t-cinnamic acid < RTI ID = 0.0 > 360 & 130 9 480 [mu] g / ml of t-cinnamic acid 176 10 m-cumaric acid < SEP > 360 < 138 11 m-cumaric acid 480 [mu] g / ml 190 12 ρ-coumaric acid 360 μg / ml 146 13 ρ-coumaric acid 480 μg / ml 167 14 Ferric acid 360 / / ml 133 15 480 < RTI ID = 0.0 > pg / ml & 159 16 360 mu g / ml pomonotetine 133 17 480 < RTI ID = 0.0 > pg / ml < 170 18 Homogenethicinic acid 360 [mu] g / ml 133 19 Homogenethicinic acid 480 [mu] g / ml 188 20 Camphorol 360 [mu] g / ml 153 21 480 [mu] g / ml of camphor ∞ 22 Myristicine 360 / / ml 133 23 Methyletin 480 [mu] g / ml 180 24 Long 360 ㎍ / ml 130 25 Naring Long 480 / / ml 176 26 360 g / ml of pyrogallol 146 27 480 / / ml of pyrogallol 188 28 beta -resolylic acid < / RTI > 360 < 150 29 480 < RTI ID = 0.0 > pg / ml < ∞ 30 Salicylic acid 360 ㎍ / ml 163 31 Salicylic acid 480 [mu] g / ml ∞

As shown in FIG. 2 and Table 3, it was found that 185% of benzoic acid, infinity of biochanin A, 176% of t-cinnamic acid, m-cumaric acid m (Homo sapiensis acid), Formononetin (Formononetin) and Homogentisic acid (Formic acid) are 190%, 167%, 159% (188%), Kaempferol is infinity, Myricetin is 180%, Naringin is 176%, Pyrogallol is 188%, β-Resorcylic acid, And infinite salicylic acid (infinite) compared to the antithrombotic activity of other phenol compounds.

It is also possible to use benzoic acid, biochanin A, t-cinnamic acid, m-Coumaric acid, ρ-Coumaric acid, Ferulic acid, Formononetin, Homogentisic acid, Kaempferol, Myricetin, Naringin, Pyrogallol, β-resole Β-Resorcylic acid and Salicylic acid were found to be comparable or superior to thrombin inhibitory activity when compared with aspirin, a positive control used as an anti-thrombotic drug.

As shown in FIG. 3 and Table 4, again, the 14 phenolic compounds showed thrombin inhibitory activity in a concentration-dependent manner. As a result, a concentration-dependent thrombin inhibitory activity pattern was confirmed. Therefore, it was confirmed that the above-mentioned 14 phenolic compounds are compounds having an antithrombotic effect.

2. From the Golden Chapters Detached  Phenolic Trombo Plastin  Measurement of inhibitory activity

Thromboplastin inhibitory activity was measured by the same method as in Example 2-2 using 50 占 퐇 of aPTT reagent (sysmex, actin) instead of thrombin. As a positive control, warfarin (Sigma Chemicla Co.) was dissolved in DMSO at a concentration of 50 mg / ml. The inhibitory activity was calculated by the formula (2).

[ Equation  2]

Active fraction thromboplastin inhibitory activity (%) = (coagulation time in sample treatment / coagulation time in negative control) x 100

The above results are shown in FIG. 4 and FIG. As shown in FIG. 4, warfarin, which is a positive control, was twice as active as DMSO, a negative control, t-Cinnamic acid was 185%, o-Coumaric acid acid is 160%, ρ-Coumaric acid is 180%, Formononetin is 185%, Homogentisic acid is 181%, Myricetin is 164% 173% for Protocatechuic acid, 188% for Pyrogallol, 163% for Sinapic acid, 184% for Syringic acid and 184% for Veratric acid ) Was 195%, confirming the relatively excellent thromboplastin inhibitory activity as compared with other phenol compounds.

As shown in Fig. 5, the thrombin inhibitory activity of t-cinnamic acid, m-cumaric acid (m- Coumaric acid, ρ-Coumaric acid, Ferulic acid, Formononetin, Homogentisic acid, Myricetin, and Pyrogallol Showed strong antioxidant activity against thromboplastin inhibition and showed that it did not coagulate in the experimental system over the positive control warfarin.

Thus, it has been confirmed that the above-mentioned eight kinds of derived phenolic compounds act even at the upper stage of the endogenous coagulation reaction and have a strong antithrombotic effect.

3 . Determination of prothrombin inhibitory activity of phenol compounds isolated from golden scales

The prothrombin inhibitory activity was measured in the same manner as in Example 2-2 using 100 占 퐇 of PT reagent (Sysmex, Thromborel S) instead of aPTT reagent. The prothrombin inhibitory activity was measured by the following equation (3). The results are shown in Figures 6 and 7.

[ Equation  3]

Prothrombin inhibitory activity (%) = (coagulation time in sample treatment / coagulation time in negative control) x 100

As shown in FIG. 6, warfarin, which is a positive control, was twice as active as DMSO, a negative control. As expected, benzoic acid was 172%, biochanin A was 203% The content of caffeic acid was 189%, that of t-cinnamic acid was 185%, that of m-Coumaric acid was 172%, that of o-Coumaric acid was 205% 201% of ρ-Coumaric acid, 201% of ferulic acid, 172% of Formononetin, 152% of Gallic acid, Homogentisic acid is 172%, camempferol is 200%, pyrogallol is 181%, beta -resorcylic acid is 171%, salicylic acid is 164% , Vanillic acid (156%), and veratric acid (155%). These compounds showed relatively good prothrombin inhibitory activity compared to other phenol compounds.

As shown in FIG. 7, the prothrombin inhibitory activity was determined in a concentration-dependent manner. As a result, it was confirmed that benzoic acid, biochanin A, t-cinnamic acid (t M-coumaric acid, p-coumaric acid, ferulic acid, formononetin, homogentisic acid, Kaempferol, Pyrogallol, β-Resorcylic acid and Salicylic acid also showed a strong activity against prothrombin inhibition and were superior to the positive control warfarin But not to coagulate.

Therefore, it has been confirmed that the above-mentioned 12 kinds of derived phenolic compounds act in the upper stage of the extrinsic coagulation reaction and have a strong antithrombotic effect.

Formulation example  1. Preparation of Pharmaceutical Compositions

1-1. Sanje  Produce

20 mg of the phenol compound of the present invention

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

10 mg of the phenol compound of the present invention

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Preparation of capsules

10 mg of the phenol compound of the present invention

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

10 mg of the phenol compound of the present invention

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _{4 ?} 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1-5. Liquid  Produce

20 mg of the phenol compound of the present invention

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  2. Formulation of food composition

2-1. Manufacture of health food

100 mg of the phenol compound of the present invention

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2-2. Health drink  Produce

100 mg of the phenol compound of the present invention

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 L container, ≪ / RTI >

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (6)

 A blood coagulation inhibitor comprising Formononetin as an active ingredient and inhibiting the activity of blood coagulation enzyme selected from the group consisting of thrombin, prothrombin and thromboplastin. 2. The anticoagulant according to claim 1, wherein the formononetin is extracted and separated from the gold wax. delete delete delete delete

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