KR20200069962A - Pharmaceutical composition comprising the extract of darae pollen 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 including pollen of Actinidia arguta as an active ingredient for preventing or treating thrombosis. Particularly, the present invention relates to a pharmaceutical composition and health-aid food including ethyl acetate fraction, butanol fraction, residue in water after fractionation of ethanol extract of pollen of Actinidia arguta, or a mixture thereof, as an active ingredient for preventing, treating/alleviating thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation. The extract of pollen of Actinidia arguta as an active ingredient of the pharmaceutical composition and health-aid food for preventing or treating thrombosis inhibits thrombogenesis-related enzymes and blood coagulation factors and also inhibits platelet aggregation functioning to initiate thrombogenesis, thereby providing strong anti-thrombotic activity. In addition, the extract of pollen of Actinidia arguta shows no hemolysis to human red blood cells, has excellent heat stability and causes no loss of the effect of inhibiting blood coagulation factors and thrombogenesis-related enzymes, and thus is expected to be used to prevent and treat thrombosis, such as ischemic stroke and hemorrhagic stroke, by improving blood circulation, even under an acidic condition of pH 2 and in the plasma. The active ingredient can be processed into various shapes, such as extract, powder, pills and tablets, and prepared into forms that can be administered at all times. Therefore, the present invention is useful in the field of pharmaceutical and food industries.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF DARAE POLLEN 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 preventing or treating thrombosis containing Pollen of Actinidia arguta extract as an active ingredient, and more specifically, ethyl acetate of Pollen ethanol extract It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibiting blood clotting and inhibiting platelet aggregation using fractions, water residues after fractionation, or mixtures thereof as an active ingredient, and health functional foods.

Blood as a component of the human body has various important functions such as transport function and buffer function of oxygen, nutrients, wastes, maintenance of body temperature, regulation of osmotic pressure and maintenance of ion balance, maintenance of moisture, fluid control, maintenance and regulation of blood pressure, and biological defense. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolytic reaction system in the body are complementarily regulated, and among these, the mechanism of the blood coagulation reaction system is that platelets adhere to and adhere to blood vessel walls to form platelet thrombus. , It has been reported that the blood coagulation system activates to form a fibrin thrombus around a platelet aggregate.

The production of fibrin thrombus is a thrombin that is involved in fibrin coagulation through several step reactions of a number of blood coagulation factors, finally producing fibrin monomer from fibrinogen, and the fibrin monomers are polymerized by calcium to platelet and endothelial cells. It binds and forms a permanent thrombus, forming a fibrin polymer cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus production, such as promoting blood clotting reactions by activating platelets, factor V and factor VII. Therefore, an active inhibitor of thrombin can be used as a very useful preventive and therapeutic agent for various thrombotic diseases caused by excessive blood clotting abnormalities. On the other hand, it is known that the activation of prothrombin following the sequential activation of factor XII, factor XI, factor IX, factor X in the endogenous thrombus production pathway finally activates thrombin. It is targeted. To date, various anticoagulants such as heparin, coumarin, aspirin, and eurokinase, antiplatelet agents, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases, but they are not only very expensive, but also have bleeding side effects, gastrointestinal disorders, and hypersensitivity reactions. The use thereof is limited.

On the other hand, Actinidia arguta is an edible or medicinal tree of the Actinidiaceae family, about 2 to 5 m tall, with broad ovate and oval leaves with thin, sharp jagged edges, and fruits reddish in July-August. Running and ripe fruits are popular because they have a similar taste to kiwi (sheep). In addition, young leaves are used as herbs, and in oriental medicine, the fruits are used for the treatment of paralysis, nephritis, liver disease, gastritis, and diabetes.

In addition, pollen is a reproductive organ of related plants that make seeds, and is a structure surrounded by a sturdy shell to protect male spouses, and is mainly carried by wind or insects to reach female spouses and fertilize to form seeds. do. The pollen is divided into insect-mediated pollen pollen and wind-mediated pollen pollen, and the impregnated pollen is divided into single plant pots such as sputum pots and acorn pots and sundries with various plant pots. Songhwabun.

Normally, pollen contains 20~25% crude protein, 7~15% crude lipid, 2~5% crude ash, 20~50% crude carbohydrate, and 10~15% moisture. It has been used as a nutritional supplement and traditional medicine. However, the pollen is composed of a thick layer of sporopollenin (extine) and an endothelial (intime), so it is not digested and decomposed by itself, so the bioavailability is low at 10-15%. There is (Pyeon HI, 2018, Journal of Life Science 28: 605 ~ 614), microbial contamination may be caused by a variety of plants, there is also a disadvantage that easily decay. In addition, toxic substances (1,2-dehydro pyrollizidine alkaloids) suspected of liver toxicity, pulmonary toxicity, and carcinogenicity have been identified in Western fern flowers (Boppre, M et al., J. Agric. Food Chem. 53, 594- 600).

Therefore, the research on the existing pollen was mainly focused on the development of extraction conditions and pretreatment methods to improve digestion and absorption by decomposing the nutritional properties of the pollen and the solid outer shell, but recently, research on useful physiological activity has been conducted. It is known to be effective against various diseases such as vascular and circulatory system, digestive system, metabolic system, and aging. Antibacterial and antioxidant effects, immune enhancement effect, prostate hyperplasia and prostatitis treatment effects have been reported (Choi et al., 2007) , Korean J. Food preserv. 14, 87-93; Abouda et al., 2011, J. Microbiol. 6, 376-384; Li et al., 2009, Carbohydrate Polymers 78, 80-88; Inpyo Hong et al., 2016, Journal of Apiculture 31, 219-225; Inpyo Hong, 2014, Journal of Apiculture 29, 137-142; Park Yong-ki et al., 2015. Journal of Apiculture 30, 299-306; Kim Seok-jung et al., 2005, Korean Journal of Food Science and Technology 37, 833-837 ).

On the other hand, studies related to thrombus in pollen have been reported to be very limited only in willow pollen and calendula, especially in the case of calendula, anti-thrombotic and hemostatic promoting activity is known. In fact, most pollen (pollen) causes allergies and is known to stimulate platelet activation factors to promote platelet aggregation (Zhou, B et al., 2011, Journal of Huazhong University of Science and Technology. 31: 589-590) . As a study related to the antithrombotic activity of willow pollen, a study on the characteristics of thrombolytic enzymes in willow pollen (Hyemin Park et al., 2009, CNU Journal of agricultural science v.36 no.1, pp. 111-122), , Hemostatic inhibitory effect (Zhao, G, 2011, Chinese journal of biochemical pharmaceutics 32: 222-224) is known, and it has been reported that the anticoagulant in the pollen is a polymer polysaccharide (Gibbs, A et al., 1983, Thrombosis research 32) : 97-108). In addition, it has been reported that silsosangamitang extract containing willow pollen inhibits platelet aggregation and fibrin aggregation (Park WH et al., 2004, Phytother Res. 18, 224-229). Therefore, it has been confirmed that the willow pollen exhibits antithrombotic activity due to thrombolytic activity, anticoagulant activity, and antiplatelet activity.

In addition, in studies related to the antithrombotic activity of pine pollen obtained from pine trees, the lipid component of calendula acts on platelets to promote agglutination reactions or inhibits platelet activating factor (PAF), a type of phospholipid that induces physiological activity. Therefore, it has been reported that it finally inhibits platelet aggregation (Siafaka-Kapadai, A et al., 1986, Biochemistry international v.12 no.1, pp. 33-41). However, it is known that the lipid component of the pine pollen promotes platelet aggregation at high concentrations and mediates different reactions depending on the concentration. Therefore, in the case of pine pollen, it exhibits antithrombotic activity or anti-thrombogenicity-promoting activity depending on [component composition and treatment concentration].

In addition, as a patent related to thrombosis of pollen, [Antithrombotic composition containing a pollen extract] targeting a thrombolytic enzyme is known from Korean Patent Registration No. 10-0913370, and the antithrombotic use of a pollen pollen in WO-4009575 [An extract of a typhae pollen and its manufacture and use], US Patent Publication No. US-0018261 discloses the effect of improving blood circulation of a pollen pollen in [Method and use of extract of a member of Typhaceae's family].

However, no research or patent literature has been known on the antithrombotic activity of sputum pollen extract and fractions thereof.

KR 10-0913370 B

Dae-Gyun Jung et al., 2007. Composition for the prevention and treatment of cardiovascular diseases, containing compounds isolated from Darae pollen extract, food technology, 20, 75-84. Jeong Sook Kwon et al., 2004, Search for Thrombin Inhibitors from Edible and Medicinal Wild Vegetables, J. Life Sci. 14, 509-513.

The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to provide a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing sputum pollen extract as an active ingredient. Is what you want.

In order to solve the problems as described above, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which contains an extract of Pollen of Actinidia arguta as an active ingredient.

The oleander pollen extract is preferably an ethyl acetate fraction obtained by sequentially fractionating ethanol extract of oleander pollen with hexene, ethyl acetate, and butanol, a water residue, or a mixture thereof.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains an extract of Pollen of Actinidia arguta as an active ingredient.

The oleander pollen extract is preferably an ethyl acetate fraction obtained by sequentially fractionating ethanol extract of oleander pollen with hexene, ethyl acetate, and butanol, a water residue, or a mixture thereof.

The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the Darae pollen extract as an active ingredient in a dietary supplement are effective in inhibiting the aggregation of platelets that serve to initiate thrombus generation together with inhibition of thrombus-related enzymes and blood clotting factors. In addition, it exhibits strong antithrombotic activity, and does not show any hemolytic activity against human red blood cells, has excellent thermal stability, and has an effect of inhibiting blood clotting factor inhibition and blood clot production-related enzyme inhibitory effects in acidic conditions and plasma at pH 2. Since it does not appear, it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredient is processed into various forms such as extract, powder, pills, tablets and can be taken at all times. It is a very useful invention in the pharmaceutical industry and the food industry because it has excellent effects that can be formulated in the form.

1 is a photograph of a potted plant.
2 is a microscopic (200 times) observation photograph of a potted plant.
Figure 3 shows the human platelet aggregation inhibitory activity of ethanol extract of Darae pollen and its sequential organic solvent fraction: 1: solvent control (DMSO), 2: aspirin (0.25 mg/ml), 3: aspirin (0.125 mg/ml) , 4: Darae pollen ethanol extract (0.25mg/ml), 5: Hexene fraction of Darae pollen ethanol extract (0.25mg/ml), 6: ethyl acetate fraction of Darae pollen ethanol extract (0.25mg/ml), 7: Darae Butanol fraction (0.25 mg/ml) of pollen ethanol extract, 8: Water residue (0.25 mg/ml) after fractionation of organic solvent of potflower ethanol extract.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, in order to test the anti-thrombotic efficacy against the sputum pollen, prepared the sputum pollen extract in a certain method, and recovered the ethyl acetate fraction and the water residue after the organic solvent fraction as an antithrombotic active ingredient, Extracts and fractions of human erythrocytes, while showing no hemolytic activity at all, by confirming that it has excellent thermal stability and acid stability characteristics of the extracts and fractions as a pharmaceutical composition and health functional food for prevention or treatment/improvement of thrombosis I wanted to utilize it.

Specifically, the present inventors develop a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis using Darae pollen, which is known to be effective in various diseases such as vascular and circulatory systems, digestive system, metabolic system, and aging in the private sector To this end, ethanol extracts were prepared for sputum pollen, and their antithrombotic activity was directly inhibited by human thrombin against human plasma and human thrombin (Thrombin Time), prothrombin time (Prothrombin Time) and active part thromboplastin time (activated) Partial Thromboplastin Time (apTTT) was evaluated to confirm that it had good anticoagulant activity, and it was found that ethyl acetate fraction obtained by sequential organic solvent fractionation and water residue after organic solvent fractionation inhibited blood coagulation factor inhibition activity and strong platelet aggregation inhibition. Activity was confirmed. In addition, it was confirmed that the extract and active fractions did not show hemolytic activity against human red blood cells.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which contains an extract of Pollen of Actinidia arguta as an active ingredient.

The oleander pollen extract is preferably an ethyl acetate fraction obtained by sequentially fractionating ethanol extract of oleander pollen with hexene, ethyl acetate, and butanol, a water residue, or a mixture thereof.

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains an extract of Pollen of Actinidia arguta as an active ingredient.

The oleander pollen extract is preferably an ethyl acetate fraction obtained by sequentially fractionating ethanol extract of oleander pollen with hexene, ethyl acetate, and butanol, a water residue, or a mixture thereof.

Hereinafter, the method and the efficacy test of the sputum pollen extract of the present invention and each active fraction will be described in more detail.

The present invention comprises the steps of preparing an extract from a potted plant; Sequential organic solvent fraction preparation of hexene, ethyl acetate, butanol from ethanol extract from Darae pollen and preparation of water residues obtained afterwards; It includes the step of evaluating the antithrombotic activity of the extracts and fractions and the stability of water residues after ethyl acetate fractions and organic solvent fractions.

The "sera pollen extract" contained in the composition of the present invention can be obtained by the step of extracting the pollen pollen with an organic solvent and filtering the extract using a filter network of 0.06 mm or less, and concentrating it under reduced pressure.

The organic solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), mixed solvents of the lower alcohol and water, etc. Can be used, hot water, or 95% ethanol extraction is most preferred.

As a preferred embodiment, the sage pollen extract of the present invention can be used by extracting sage pollen with hot water or ethanol. Here, the hot water or ethanol extract may be sequentially or separately fractionated with an organic solvent of hexene, ethyl acetate, and butanol to additionally obtain a hexene fraction, ethyl acetate fraction, butanol fraction, and water residue.

In the present invention, as a result of measuring thrombin time, prothrombin time, and apity time at a concentration of 5 mg/ml of Darae pollen extract, it showed weaker anticoagulant activity than aspirin (1.5 mg/ml) known as an antithrombotic agent. Ethanol extract showed better blood clotting inhibitory activity than hot water extract. However, as a result of confirming the inhibition of platelet aggregation with a concentration of 0.25 mg/ml of Darae pollen extract, it showed 81% aggregation rate in the hot water extract and 19% aggregation inhibition, whereas the ethanol extract showed aggregation of 134.7%, resulting in platelet aggregation. It showed palpation. However, in the case of the hot water extract, the total sugar and reducing sugar content is too high, which makes it difficult to further refine the ethanol extract, sequentially fractionating the organic solvent and evaluating the antithrombotic activity of each fraction.

As a result, in the case of ethyl acetate fraction, butanol fraction, and water residue of ethanol extract of Darae pollen, it showed 1.20~1.32 times the blood coagulation factor inhibition (aPTT) compared to no additives at a concentration of 5 mg/ml, especially the ethyl acetate fraction was 0.25 At a concentration of mg/ml, aspirin showed a strong inhibition of human platelet aggregation corresponding to 0.125 mg/ml, and a platelet aggregation inhibition of 23.1% was observed even at 0.25 mg/ml of water residue. These results indicate that the ethyl acetate fraction and water residue of Darae pollen ethanol extract have very strong antithrombotic activity, suggesting that anticoagulants such as aspirin and antiplatelet agents, which are highly concerned about side effects, can be replaced.

The sputum pollen extract of the present invention and its active fraction materials can be prepared as a powder through a conventional powdering process such as vacuum drying and freeze drying, or spray drying. They are not degraded by various degrading enzymes in plasma, and they maintain activity even at a heat treatment of 100°C and a pH in the human stomach of pH 2.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, sensory abnormalities, personality changes, decreased vision, epilepsy attacks, pulmonary thrombosis , Deep vein thrombosis, swelling of the lower extremities, pain and acute peripheral artery occlusion, and intravenous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, brain venous sinus thrombosis and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention is an oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, injection of sterile injectable solution according to a conventional method according to each purpose of use It can be formulated and used in various forms, such as oral administration or intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

The pharmaceutical composition may further include a carrier, excipient or diluent, and examples of suitable carrier, excipient or diluent that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil 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, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., such solid preparations comprising at least one excipient in the pharmaceutical composition, for example starch, calcium carbonate, It is formulated by mixing sucrose, lactose and gelatin. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

As a preferred embodiment, a liquid preparation for oral use may be exemplified by suspending agents, solvent solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, Fragrances, preservatives, and the like may be included.

As a preferred embodiment, the preparation for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections 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, "a 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 is the type of patient's disease, severity, activity of the drug, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-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, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and weight, and generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg daily Or it can be administered every other day or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., so that the dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

In the present invention, "administration" means providing a predetermined substance to a patient in any suitable way, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells.

“Subject” in the present invention is not particularly limited, and includes, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, rabbit or guinea pig. And, preferably, a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in a variety of foods and beverages effective in preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, and dietary supplements, and may be used in the form of powders, granules, tablets, capsules, or beverages. .

The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

The dietary supplement of the present invention may contain, as an essential ingredient in the indicated proportions, the above compound as an essential ingredient, and may contain, as an additional ingredient, food additives, such as natural carbohydrates and various flavoring additives.

Examples of the natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and sugars such as xylitol, sorbitol, and erythritol, and common sugars such as dextrin and cyclodextrin.

The flavoring agent may include taumatin; Natural flavoring agents such as stevia such as rebaudioside A or glycyrrhizine, and synthetic flavoring agents such as saccharin and aspartame can be used. 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 includes various nutrients, vitamins, minerals, synthetic flavoring agents, flavoring agents such as natural flavoring agents, coloring agents and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloids It may contain a thickening agent, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonic acid used in carbonated beverages and the like. In addition, the health functional food of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These ingredients can be used independently or in combination. The ratio of these additives is generally selected from 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 through examples. The following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

[Example]

Example 1: Preparation of sputum pollen extract and analysis of useful components thereof

In 2018, Darae pollen harvested in Andong, Gyeongbuk, was purchased and used to prepare hot water and ethanol extracts. Specifically, ethanol extract was prepared by adding 10 times ethanol to the pollen pollen sample, extracting it twice at room temperature, collecting the extract, filtering it, and then concentrating it under reduced pressure to prepare a powder, and in the case of hot pot extract of Darae pollen 10 times sterile distilled water was added to the pollen sample, repeated extraction at 100° C. for 1 hour twice, followed by filtration and concentration as described above.

The darae pollen used in the experiment was yellow to gray granules with a lightness of 48.27, a redness of 3.68, and a yellowness of 18.84, showing pH 5.6, brix 40.0, and acidity of 0.68, indicating a slightly sour taste. The moisture content was 5.8%, and it was in a very dry state, where it was able to absorb moisture again when left at room temperature for a long time (FIG. 1, Table 1). As a result of observation under a microscope (200 times), the sputum pollen is an isolated subprolate, and a dividing line, such as a barley structure of barley, is identified on the surface (FIG. 2).

[Table 1] Physicochemical Characteristics and Color Difference Analysis of Darae Pollen

As a result of analysis of nutritional components, crude protein showed 35.6%, crude fat, 8.1%, and crude carbohydrate 48.3%, which was excellent at 280kcal/100g, and the ash content was 2.2%, which included various minerals in large quantities (Table 2).

[Table 2] Analysis of Nutritional Components of Darae Pollen

Total polyphenols, total flavonoids, total sugars, and reducing sugars were measured by analyzing the components of Darae pollen extract. The total polyphenol content was 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ saturated solution in 400 μl of the extracted sample solution, and allowed to stand at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol stirring for 18 hours, 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample, 40 μl of 1 N NaOH was added again, and the absorbance at 420 nm was measured after reacting at 37° C. for 1 hour. Rutin was used as a standard reagent. The reducing sugar was quantified using the DNS method and the total sugar using the phenol-sulfuric acid method.

[Table 3] Analysis of extraction efficiency and useful components of ethanol and hot water extract from Darae pollen

As shown in Table 3, the ethanol extraction efficiency of Darae pollen was 38.5%, which was lower than the hot water extraction efficiency (54.4%), but the total polyphenol content of the ethanol extract was 2.7 times higher than that of the hot water extract, and the total sugar and reducing sugar content was hot water. The extract was 6.2 times and 10.1 times higher than the ethanol extract, and it was found that most of the hot water extract was composed of sugar. On the other hand, the total flavonoid content was similar in hot water extract and ethanol extract.

Example 2: Evaluation of blood coagulation inhibitory activity of Darae pollen extract

The blood clotting inhibitory activity of the hot water and ethanol extract of Example 1 was evaluated, and the results are shown in Table 4. At this time, the method for evaluating blood clotting inhibitory activity of Darae pollen extract was evaluated according to the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and affinity time were measured. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China). Thrombin time, prothrombin time and aptitude measurement were performed as follows.

Thrombin Time

50 μl of 0.5U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ and 10 μl of sample extract of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) at 37° C. for 2 minutes, and then 100 μl of plasma was added. After that, the time until plasma clot was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of a sample as a solvent control. In the case of DMSO, a solidification time of 32.1 seconds was shown. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the thrombin inhibitory activity was expressed as the value of the solidification time when the sample was added divided by the solidification time of the solvent control.

Prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations are added to a tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, then 130 μl of PT reagent is added and plasma is coagulated. The time until it was expressed as the average value of the experiment repeated 3 times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of a sample as a solvent control. In the case of DMSO, a solidification time of 18.1 seconds was shown. The prothrombin inhibitory activity was expressed as a value obtained by dividing the coagulation time when the sample was added by the coagulation time of the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of sample extract of various concentrations are added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) is added and again at 37° C. 3 Incubated for a minute. Then, after adding 50 μl CaCl ₂ (35 mM), the time until the plasma was clot was measured. DMSO was used as a solvent control instead of a sample, and in this case, a coagulation time of 55.1 seconds was exhibited. The result of aPTT was expressed as the average value of the experiment repeated 3 times, and the blood coagulation factor inhibitory activity was expressed by dividing the aPTT at the time of sample addition by the aPTT of the solvent control.

[Table 4] Blood coagulation inhibitory activity of Darae pollen hot water extract and ethanol extract

As a result, the ethanol extract exhibited weak blood coagulation inhibitory activity at a concentration of 5 mg/ml, and both the hot water extract and the ethanol extract exhibited prolonged aPTT due to inhibition of the blood coagulation factor. However, this inhibition was weak compared to aspirin used as an antithrombotic agent in clinical practice.

Example 3: Platelet aggregation inhibitory activity of Darae pollen extract

Human platelet aggregation inhibitory activity of the hot water extract and ethanol extract of Example 1 was evaluated, and the results are shown in Table 5. Platelets are discoid-shaped small cells that circulate blood vessels with various blood cells, and instead of nucleus, they have cytoplasmic granules containing high concentrations of various substances related to vascular damage protection and platelet aggregation, and aggregation when damage to the vascular lining appears It is an important cell that initiates thrombosis by secreting factors and forming a primary hemostatic plug in combination with collagen exposed by damage to endothelial cells. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus production. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Human platelets were used as platelets, which were 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, after resuspending in suspending buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 0.49mM MgCl ₂ , 0.25% gelatin, pH 7.4), 10 min at 3,000rpm After centrifugation, the suspension was re-suspended in a suspending buffer, and the platelet count was adjusted to be 4x10 ⁹ /ml. Thereafter, 2.5 μl collagen was added to the 1 ml suspension to react for 5 minutes, and platelet aggregation was measured at 37° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 5] Platelet aggregation inhibitory activity of Darae pollen hot water extract and ethanol extract

As shown in Table 5, first, aspirin showed 26.1% aggregation at a concentration of 0.25 mg/ml, and 45.4% aggregation at a concentration of 0.125 mg/ml, showing concentration-dependent strong platelet aggregation inhibition, and used as an antithrombotic agent in clinical practice. I was able to know the basis. On the other hand, Darae pollen hot water extract showed an aggregation degree of 81.0% at 0.25 mg/ml, and thus showed excellent aggregation inhibitory activity. On the other hand, Darae pollen ethanol extract promoted human platelet aggregation at a concentration of 0.25 mg/ml, and showed an aggregation degree of 134.7% compared to the non-added cells. Therefore, it was judged that Darae pollen contains various anti-thrombotic components and thrombogenicity-promoting components simultaneously.

Example 4: Preparation of sequential organic solvent fractions of ethanol extract of Darae flower pot and analysis of useful components thereof

The ethanol extract prepared from Example 1 was subjected to sequential organic solvent fractionation with hexene, ethyl acetate, and butanol, and finally the water residue was recovered. Table 6 shows the fractional efficiency and the component analysis results.

[Table 6] Analysis of yield and useful components of organic solvent fraction of Darae pollen ethanol extract

As shown in Table 6, the largest portion of ethanol extract from Darae pollen was transferred to the butanol fraction (48.7%), and the water residue accounted for 39.7% of the extract. The ethyl acetate fraction showing the smallest yield showed 2.6% of the extract of the pollen pollen, and it was possible to recover 1 g per 100 g of the pollen pollen. Meanwhile, as a result of analyzing the total polyphenol and total flavonoid content, the highest content of polyphenol content (208.0 mg/g) and flavonoid content (55.5 mg/g) in the ethyl acetate fraction was shown. This was a 260-fold higher polyphenol content and a 61.6-fold higher flavonoid content compared to the water residue. Therefore, it was expected that the ethyl acetate fraction of Darae pollen ethanol extract would exhibit various physiological activities.

Example 5: Anticoagulant activity of sequential organic solvent fraction of Darae pollen ethanol extract

The anticoagulant activity of the sequential organic solvent fraction of the Darae pollen ethanol extract prepared in Example 4 was evaluated in the same manner as in Example 2, and the results are shown in Table 7.

[Table 7] Anticoagulant activity of sequential organic solvent fraction of Darae pollen ethanol extract

Among the fractions of the ethanol extract of Darae pollen, the ethyl acetate fraction, butanol fraction and water residue slightly prolonged prothrombin time and apity time, and it was confirmed that they particularly inhibited blood coagulation factors that contribute to the production of endogenous thrombus. . Therefore, it was confirmed that the ethyl acetate fraction, butanol fraction, and water residue of the Darae pollen extract exhibit anticoagulant activity.

Example 6: Human platelet aggregation inhibitory activity of sequential organic solvent fractions of Darae pollen ethanol extract

Human platelet aggregation inhibitory activity of the sequential organic solvent fraction of the Darae pollen ethanol extract prepared in Example 4 was evaluated in the same manner as in Example 3, and the results are shown in Table 8.

[Table 8] Platelet aggregation inhibitory activity of sequential organic solvent fraction of Darae pollen ethanol extract

As shown in Table 8, the ethyl acetate fraction of Darae pollen strongly inhibited human platelet aggregation, and showed aggregation inhibition similar to aspirin at a concentration of 0.2525/ml at 0.125mg/ml. In addition, the water residue was weaker than the ethyl acetate fraction, but still exhibited effective blood platelet aggregation inhibitory activity. Therefore, the ethyl acetate fraction and water residue are thought to be able to supplement and replace existing antithrombotic agents, such as aspirin, whose use is limited due to hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

Example 7: Human erythrocyte hemolysis activity of Darae pollen ethanol extract and its fractions

Darae pollen is a food that has been secured by being registered as a food ingredient. To evaluate the acute toxicity potential of Darae pollen ethanol extract and its fractions, human erythrocyte hemolysis activity was evaluated, and the results are shown in Table 9. At this time, the hemolytic activity was evaluated according to an existing report (Hohn Son, 2014 ㆍKorean J. Microbiol. Biotechnol. 42: 285-292), and simply 100 μl of human red blood cells washed three times with PBS in a 96-well microplate. After adding 100 μl of sample solution of various concentrations and reacting for 30 minutes at 37° C., the reaction solution was centrifuged for 10 minutes (1,500 rpm) to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin outflow due to hemolysis was 414 nm. It was measured at. DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 9] Human erythrocyte hemolysis activity of Darae pollen extract and its fractions

First, DMSO and water used as controls had no hemolytic activity, and triton X-100 was confirmed to hemolytically erythrocyte 100% at a concentration of 1 mg/ml. In addition, in the case of amphotericin B, which is used as an anti-cancer agent and anti-fungal agent, it was confirmed that hemolysis of 50% of red blood cells was carried out at a concentration of 0.00625 mg/ml. The ethanol extract of Darae pollen of the present invention was found to have no acute toxicity and erythrocyte hemolytic activity since no erythrocyte hemolysis occurred in fractions except the hexene fraction at a concentration of 1 mg/ml. The hexene fraction showed red blood cell hemolytic activity of 97.2% at a concentration of 1 mg/ml, 52.6% at 0.5 mg/ml, and 10.5% at 0.25 mg/ml, and thus was expected to have acute toxicity potential.

Example 8: Plasma pollutant ethanol extract and its ethyl acetate fraction and water residue evaluation of plasma, acid and thermal stability

Plasma stability, thermal stability, and acid stability for antithrombotic activity were confirmed for the ethanol extract of Darae pollen obtained in Example 1 and its ethyl acetate fraction and water residue obtained in Example 4. The ethanol extract of Darae pollen and its active fractions did not show a decrease in blood clotting inhibition and platelet aggregation inhibitory activity even after 1 hour heat treatment at 100°C, 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma. , Rather, the antithrombotic activity was partially increased. Therefore, it was confirmed that the Darae pollen extract contains antithrombotic active substances of various components having acid resistance and heat resistance.

Claims (4)

A pharmaceutical composition for preventing or treating thrombosis, which contains an extract of Pollen of Actinidia arguta as an active ingredient. The pharmaceutical composition according to claim 1, wherein the sputum pollen extract is an ethyl acetate fraction, a water residue, or a mixture thereof obtained by sequentially fractionating a sage pollen ethanol extract with hexene, ethyl acetate, and butanol. Health functional food for prevention or improvement of thrombosis, which contains Pollen of Actinidia arguta extract as an active ingredient. The health functional food according to claim 3, wherein the sputum pollen extract is an ethyl acetate fraction, a water residue or a mixture thereof obtained by sequentially fractionating a sage pollen ethanol extract with hexene, ethyl acetate and butanol.

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