KR20200069965A - Pharmaceutical composition comprising the extract of acorn 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 a health functional food for preventing or treating thrombosis containing an acorn pollen (Pollen of Quercus sp.) extract as an active ingredient, and more specifically, an acorn pollen ethanol extract, its It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibiting blood clotting and inhibiting platelet aggregation by using ethyl acetate fraction, butanol fraction, water residue after organic solvent fraction, or a mixture thereof as an active ingredient and a health functional food. . The pharmaceutical composition for preventing or treating thrombosis of the present invention and the acorn 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.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF ACORN 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 an acorn pollen (Pollen of Quercus sp.) extract as an active ingredient, and more specifically, an acorn pollen ethanol extract, its It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibiting blood clotting and inhibiting platelet aggregation by using ethyl acetate fraction, butanol fraction, water residue after organic solvent fraction, or a mixture thereof as an active ingredient and a health functional food. .

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, acorn (acorn) is a generic term for a tree fruit belonging to the oak family, and more specifically, oak ( Quercus acutissima ), oak ( Quercus variabilis ), stone oak ( Quercus mongolica ), oak ( Quercus aliena ), sol Refers to oak fruits such as oak ( Quercus serrata ), water oak, and oak ( Quercus dentata ), and the pots produced by bees from the flowers of these oak plants are called acorn pots. Acorn pollen, like other Chungmae pollen, is a mixture of pollen (pollen), honey, and enzyme by worker bees, and is agglomerated like a dumpling, and humans have long used it as a health food.

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 imperial pollen is divided into single plant pots such as acorn pots, 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 on various components and efficacy studies of the fruit (acorn) and leaves of the oak plant have been extensively conducted, but studies related to acorn pollen have been almost absent. Studies related to acorn pollen include changes in ingredients by physical treatment of acorn pollen (pollen) (Hong In-pyo, 2013. Korean J. Apiculture 28, 217-221), antibacterial activity of acorn pollen germination solution (Choi Jun-hyuk, 2007, Korean food storage Distribution Journal, 14, 87-93) and antioxidant activity (Hong In-Pyo, 2014, Journal of Apiculture 29, 137-142) are known, and other activities related to acorn pollen are unknown.

In addition, as a patent related to acorn pollen, Korean Patent No. 10-1237215 [Pharmaceutical composition for preventing or treating enteritis] is known, and other patents related to physiological activity are not known.

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 on antithrombotic activity of acorn pollen extract and fractions thereof has been known to date.

KR 10-0913370 B KR 10-1237215 B

Yuk Keun-jung, Ewha Womans University Graduate School, 2001, Domestic Master's, Chestnut, Acorn's pulp and endothelial effect on fat metabolism, antioxidant and antithrombogenic activity in rats.

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 and a health functional food for preventing or treating thrombosis containing acorn pollen extract as an active ingredient. Is what you want.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which contains an extract of acorn pollen (Pollen of Quercus sp.) as an active ingredient.

The acorn pollen extract is preferably an acorn pollen ethanol extract.

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

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains an acorn pollen (Pollen of Quercus sp.) extract as an active ingredient.

The acorn pollen extract is preferably an acorn pollen ethanol extract.

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

The pharmaceutical composition for preventing or treating thrombosis of the present invention and the acorn 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 an acorn pollen.
Figure 2 is a microscope (200 times) observation picture of the acorn pollen.
Figure 3 shows the human platelet aggregation inhibitory activity of acorn pollen ethanol extract and its sequential organic solvent fraction: 1: solvent control (DMSO), 2: aspirin (0.25mg/ml), 3: aspirin (0.125mg/ml) , 4: Acorn pollen ethanol extract (0.25mg/ml), 5: Acorn pollen ethanol extract hexene fraction (0.25mg/ml), 6: Acorn pollen ethanol extract ethyl acetate fraction (0.25mg/ml), 7: Acorn Butanol fraction (0.25 mg/ml) of pollen ethanol extract, 8: Water residue (0.25 mg/ml) after fractionation of organic solvent of acorn pollen ethanol extract.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared an acorn pollen extract in a certain way to test the antithrombogenic efficacy of the acorn pollen, and a water residue after the acorn pollen ethanol extract, its ethyl acetate fraction, butanol fraction and organic solvent fraction It was recovered as a thrombus active ingredient, and the extracts and fractions were used for the prevention or treatment/improvement of the thrombosis by confirming that the extracts and fractions have excellent thermal stability and acid stability without showing any hemolytic activity against human red blood cells. It was intended to be used as a pharmaceutical composition and dietary supplement.

Specifically, the present inventors developed a pharmaceutical composition for preventing or treating/improving thrombosis using acorn pollen known to be effective in various diseases such as blood vessels and circulatory system, digestive system, metabolic system, and aging in the private sector and developing health functional foods To this end, ethanol extracts were prepared for acorn pollen, and their antithrombotic activity was directly inhibited by human thrombin against human plasma and human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin Time) and active part thromboplastin time (activated) Partial Thromboplastin Time (apTTT) was evaluated to confirm good anticoagulant activity, and strong platelet aggregation inhibitory activity was confirmed in ethyl acetate fraction obtained by sequential organic solvent fractionation, butanol fraction, and water residue after organic solvent fractionation. Did. 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 acorn pollen (Pollen of Quercus sp.) as an active ingredient.

The acorn pollen extract is preferably an acorn pollen ethanol extract.

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

In addition, the present invention provides a health functional food for preventing or improving thrombosis, which contains an acorn pollen (Pollen of Quercus sp.) extract as an active ingredient.

The acorn pollen extract is preferably an acorn pollen ethanol extract.

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

Hereinafter, a method and an efficacy test of the acorn 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 acorn pollen; Sequential organic solvent fraction preparation of hexene, ethyl acetate, butanol from acorn pollen ethanol extract and preparation of water residues subsequently obtained; Evaluating antithrombotic activity of the extract and fraction; And ethanol extract, its ethyl acetate fraction, butanol fraction, and organic solvent fraction after the stability of the water residue.

The "acorn pollen extract" contained in the composition of the present invention can be obtained by extracting the acorn 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 acorn pollen extract of the present invention can be used by extracting the acorn 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 with acorn pollen extract at a concentration of 5 mg/ml, 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. In addition, as a result of confirming the inhibition of platelet aggregation by using acorn pollen extract at a concentration of 0.25 mg/ml, aggregation inhibition was not observed due to a 105.5% aggregation rate in the hot water extract, whereas aggregation of 81.4% in the ethanol extract showed aggregation activity. Was confirmed. Therefore, sequential organic solvent fractions were prepared for the ethanol extract of acorn pollen, and the antithrombotic activity of each fraction was evaluated.

As a result, in the case of the ethyl acetate fraction of the acorn pollen ethanol extract, it was confirmed that the thrombin time, prothrombin time and apity time were extended by 1.12 times, 1.21 times, and 1.16 times compared to the additive-free group at a concentration of 5 mg/ml. In addition, the ethyl acetate fraction, butanol fraction and water residue showed a platelet aggregation degree of 48.3 to 59.1% compared to the additive-free group at a concentration of 0.25 mg/ml, indicating a strong inhibition of human platelet aggregation corresponding to 0.125 mg/ml of aspirin. These results indicate that the ethyl acetate fraction of acorn pollen ethanol extract has very strong antithrombotic activity, and suggests that anticoagulants such as aspirin and antiplatelet agents, which are highly concerned about side effects, can be substituted.

The acorn pollen extract of the present invention and its active fraction materials may be prepared as a powder through a conventional powdering process such as vacuum drying and lyophilization, 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 acorn pollen extract and analysis of useful components thereof

In 2018, acorn pollen harvested in Korea was purchased and used to prepare hot water and ethanol extracts. Specifically, 10 times ethanol was added to the sample of acorn pollen, repeated extraction at room temperature twice, the extract was collected, filtered, and concentrated under reduced pressure to prepare a powder to produce an ethanol extract. In the case of acorn pollen hot water extract, acorn 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 acorn pollen used in the experiment was dark yellow granules with a brightness of 40.18, a redness of 3.58, and a yellowness of 20.29, indicating a more vivid yellow color than Darae pollen produced in Korea. In addition, the acorn pollen exhibited pH 4.6, brix 52.0, and acidity 0.74, which showed higher sugar content and acidity than that of Darae pollen pH 5.6, brix 40.0, and acidity 0.68. The moisture content was 11.5% in a very dry state, and was allowed 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 acorn pollen was identified as an isolated prolate, the polar surface was circular, and the surface was confirmed to be granular or granular (Fig. 2).

[Table 1] Physicochemical properties and color difference analysis of acorn pollen

As a result of nutritional component analysis, crude protein 25.1%, crude fat, 12.5%, crude carbohydrate showed 48.0%, which was excellent at 404.9 kcal/100g, and the ash content also showed 1.9%, which contained various minerals in large quantities (Table 2). It can be seen that it contains more crude fat when compared to 35.6%, crude fat, 8.1%, and 48.3% crude carbohydrate content of Darae pollen.

[Table 2] Analysis of Nutritional Components of Acorn Pollen

The content of total polyphenol, total flavonoid, total sugar and reducing sugar was measured by analyzing the components of the acorn 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 acorn pollen ethanol and hot water extract

As shown in Table 3, the extraction efficiency of the acorn pollen ethanol extract was 46.4%, which was lower than that of the hot water extract (56.5%), but the total polyphenol content of the ethanol extract was 1.5 times higher than the hot water extract, and the total sugar and The reducing sugar content of the hot water extract was 1.3 times and 1.36 times higher than that of the ethanol extract. On the other hand, the total flavonoid content was similar in hot water extract and ethanol extract.

Example 2: Evaluation of blood clotting inhibitory activity of acorn 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 the blood clotting inhibitory activity of acorn 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 clotting inhibitory activity of acorn pollen hot water extract and ethanol extract

As a result, the ethanol extract exhibited a weak blood clotting inhibitory activity at a concentration of 5 mg/ml, and the thrombin time by thrombin inhibition was extended 1,12 times compared to the additive-free group. However, this inhibition was weak compared to aspirin used as an antithrombotic agent in clinical practice.

Example 3: Platelet aggregation inhibitory activity of acorn 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 acorn pollen hot water extract and ethanol extract

As shown in Table 5, 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, which is the basis for clinical use as an antithrombotic agent. Was able to know. On the other hand, acorn pollen hot water extract showed an aggregation degree of 105.5% at 0.25 mg/ml, and thus did not affect aggregation, and the ethanol extract showed an aggregation degree of 81.4%, showing excellent aggregation inhibitory activity.

Example 4: Preparation of sequential organic solvent fractions of acorn pollen ethanol extract and analysis of useful components thereof

The acorn pollen ethanol extract prepared in 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 acorn pollen ethanol extract

As shown in Table 6, the largest portion of the acorn pollen extract was transferred to the butanol fraction (64.8%), and the water residue accounted for 25.1% of the extract. The ethyl acetate fraction showing the smallest yield showed 4.9% of the acorn pollen extract, and 2.27 g per 100 g of acorn pollen could be recovered. On the other hand, as a result of analyzing the total polyphenol and total flavonoid content, the highest content of polyphenol content (225.0 mg/g) and flavonoid content (222.1 mg/g) in the ethyl acetate fraction was shown. It was 2,250 times higher polyphenol content and 158.6 times higher flavonoid content than the lowest water residue. Therefore, the ethyl acetate fraction of the acorn pollen extract was expected to exhibit a variety of physiological activities.

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

The anticoagulant activity of the sequential organic solvent fraction of the acorn 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 acorn pollen ethanol extract

Among the fractions of the acorn pollen extract, the ethyl acetate fraction exhibited good antithrombotic activity by extending thrombin time, prothrombin time and apity time by 1.12, 1,21 and 1,16 times compared to no additives.

Example 6: Human platelet aggregation inhibitory activity of sequential organic solvent fraction of acorn pollen ethanol extract

Human platelet aggregation inhibitory activity of the sequential organic solvent fraction of the acorn 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 acorn pollen ethanol extract

As shown in Table 8, the ethyl acetate fraction, butanol fraction, and water residue of the acorn pollen ethanol extract exhibited human platelet aggregation of 48.3 to 59.1% at a concentration of 0.25 mg/ml, platelet aggregation similar to aspirin 0.125 mg/ml concentration. It showed inhibitory activity. Therefore, it is believed that the active fraction of acorn pollen can supplement and replace existing antithrombotic agents such as aspirin, which are restricted in use due to hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

Example 7: Human erythrocyte hemolysis activity of acorn pollen ethanol extract and fractions thereof

Acorn pollen is food that has been secured by being registered as a food ingredient. To evaluate the acute toxicity potential of acorn 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 red blood cell hemolytic activity of acorn 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. It was confirmed that the fractions except the hexene fraction of the acorn pollen ethanol extract of the present invention have no acute toxicity and red blood cell hemolysis activity because no red blood cell hemolysis occurred up to a concentration of 1 mg/ml. The hexene fraction exhibited red blood cell hemolytic activity of 89.9% at a concentration of 1 mg/ml, 53.5% at 0.5 mg/ml, and 7.5% at 0.25 mg/ml, and thus was expected to have acute toxicity potential.

Example 8: Evaluation of plasma, acid and thermal stability of acorn pollen ethanol extract and active fractions thereof

Plasma stability, thermal stability, and acid stability against antithrombotic activity were confirmed for the acorn pollen ethanol extract obtained in Example 1 and ethyl acetate fraction, butanol fraction, and water residue obtained in Example 4. Acorn pollen ethanol extract 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 acorn 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 acorn pollen (Pollen of Quercus sp.) extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the acorn pollen extract is an acorn pollen ethanol extract. The method according to claim 2, wherein the acorn pollen ethanol extract is an ethyl acetate fraction obtained by sequentially fractionating acorn pollen ethanol extract with hexene, ethyl acetate and butanol, a butanol fraction, a water residue or a mixture thereof. Composition. A health functional food for preventing or improving thrombosis comprising the active ingredient according to any one of claims 1 to 3.

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