KR102216221B1 - Pharmaceutical composition comprising the extract of pine 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 functional food for the prevention or treatment of thrombosis containing a pine tree extract as an active ingredient, and more specifically, an ethyl acetate fraction of an ethanol extract of pine tree flour, It relates to a pharmaceutical composition for preventing or treating/improving thrombosis by inhibiting blood coagulation and inhibiting platelet aggregation containing a butanol fraction, a water residue after an organic solvent fraction, or a mixture thereof as an active ingredient, and a health functional food. Songhwamin extract as an active ingredient of a pharmaceutical composition for preventing or treating thrombosis and a health functional food of the present invention is due to the inhibitory effect of platelet aggregation, which plays a role in initiating thrombus formation along with inhibition of blood clotting-related enzymes and blood clotting factors. At the same time, it exhibits strong antithrombotic activity, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, and shows a loss of blood coagulation factor inhibitory effect and blood clot formation-related enzyme inhibitory effect even in acidic conditions of pH 2 and plasma. Therefore, it is expected that it can 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, etc. It is a very useful invention in the pharmaceutical industry and food industry because it has an excellent effect that can be prepared with.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing pine pollen extract as an active ingredient {PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACT OF PINE 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 health functional food for the prevention or treatment of thrombosis containing a pine tree extract as an active ingredient, and more specifically, an ethyl acetate fraction of an ethanol extract of pine tree flour, It relates to a pharmaceutical composition for preventing or treating/improving thrombosis by inhibiting blood coagulation and inhibiting platelet aggregation containing a butanol fraction, a water residue after an 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 transporting and buffering oxygen, nutrients and waste products, maintaining body temperature, maintaining osmotic pressure and maintaining ionic equilibrium, maintaining moisture schedule, controlling liquid properties, maintaining and controlling blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombosis dissolution system are complementarily regulated in the body. Among them, the mechanism of the blood coagulation reaction system is after platelets adhere and aggregate on the vessel wall to form platelet thrombi. , It has been reported that the blood coagulation system is activated and fibrin thrombus is formed around the platelet aggregate.

The formation of fibrin thrombi leads to activation of thrombin, which is involved in fibrin coagulation, through several step reactions of numerous blood coagulation factors. Finally, fibrin monomers are produced from fibrinogen, and fibrin monomers are polymerized by calcium, resulting in platelets and endothelial cells. It binds and forms a fibrin polymer that is cross-linked by factor XIII, creating a permanent blood clot. In addition, thrombin plays a central role in the formation of blood clots by activating platelets, factors V, and VII to promote blood clotting reactions. Therefore, the thrombin activity inhibitory substance can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood coagulation. On the other hand, in the endogenous thrombogenic pathway, the sequential activation of factor XII, factor XI, factor IX, and factor X, followed by prothrombin activation, is known to ultimately activate thrombin, and specific inhibition of blood coagulation factors is also important. Being a target. To date, various anticoagulants such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases, but they are not only very expensive, but also bleeding side effects, gastrointestinal disorders and irritability. The situation is limited to such use.

On the other hand, pine ( Pinus densiflora Siebold or other fauna and flora) is an upper tree coniferous arboreous tree of the pine family, and is called pine, pine, and alder tree in Korea. Has become. The seeds of pine trees are 5~6mm long and 3mm wide, oval, black brown, and the wings have black brown stripes on a light brown background, and the leaves are used as a treatment or tonic for indigestion, flowers for dysentery, and rosin as a raw material for plaster. Flowerpots were used to make tea and traditional alcoholic beverages, while the skin was used to make Songgidduk.

Pollen is a reproductive organ of a vascular genus that makes seeds. It is a structure surrounded by a sturdy shell to protect the male gamete, and it is transported by wind or insects to reach the female gamete and forms seeds after fertilization. Pollen is divided into chungmae pots mediated by insects and air pollen pots mediated by wind, and chungmae pots are divided into single plant pots such as acorn pots and acorn pots, and miscellaneous pots in which various plant pots are collected. Can be lifted.

In general, pollen contains 20 to 25% crude protein, 7 to 15% crude lipid, 2 to 5% crude ash, 20 to 50% crude carbohydrate, and 10 to 15% moisture, so it is excellent in nutrition and contains various physiologically active substances. It has been used as a nutritional supplement and traditional medicine. However, pollen is composed of a thick layer of sporopollenin (extine) and endothelium (intime), so it cannot be digested or decomposed by itself, so the bioavailability is low, 10-15%. (Pyeon HI, 2018, Journal of Life Science 28: 605~614), microbiological contamination may appear due to various plants, and there is also a disadvantage of easily decaying. In addition, toxic substances (1,2-dehydro pyrollizidine alkaloids) suspected of hepatotoxicity, lung toxicity, and carcinogenicity have been identified in pollen of Western borer flowers (Boppre, M et al., J. Agric. Food Chem. 53, 594- 600).

Therefore, the existing research on pollen has been mainly focused on the development of extraction conditions and pretreatment methods for enhancing digestion and absorption by decomposing the nutrient properties of pollen and its solid outer shell, but recently, studies on useful physiological activities have been conducted. It is known to be effective in various diseases such as vascular and circulatory system, digestive system, metabolic system, and aging, and antibacterial and antioxidant effects, immunity enhancing effects, prostatic hypertrophy 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; Hong et al., 2016, Journal of Apiculture 31, 219-225; In-Pyo Hong, 2014, Journal of Apiculture 29, 137-142; Yong-Gi Park et al., 2015. Journal of Apiculture 30, 299-306; Seok-Joong Kim et al., 2005, Korean Journal of Food Science 37, 833-837 ).

Songhwabun is called Songhwa or Songhwa. In Korea, it is a pale yellow pine pollen collected only for 10 days from April to May. It contains a large amount of useful nutrients for the human body and has been used in the manufacture of Dasik and Songhwaju since ancient times. In addition, in the private sector, it has been applied as a treatment for diabetes and fatty liver and as a hemostatic agent.In oriental medicine, it is used to treat tonic, gangjeong and neurological disorders, heart disease, acute chronic prostatitis, hyperlipidemia, arteriosclerosis and anemia. When you eat it, it is gyeongshin (經身), and its efficacy is recorded as superior to pine, pine, and fruit.

The main ingredients of pine pollen are 66% crude carbohydrate, 15% crude protein, 5% crude lipid, and 3.2% crude ash (Lee Young-ju, 1994, J. Korean Soc. Food Nutr. 23, 192-197). It is slightly different from 20-50%, crude protein 20-25%, crude lipid 7-15%, crude flour 2-5%, moisture 10-15%, and is characterized by high carbohydrate content but low crude lipid content. .

Pollen thrombosis-related studies have been reported very limited only in caterpillar pollen and pine pollen, but most pollen (pollen) are known to induce allergies and stimulate platelet activation factors to promote platelet aggregation (Zhou, B, etc., 2011, Journal of Huazhong University of Science and Technology. 31: 589-590).

As a study on antithrombotic activity of caterpillars pollen, a study on the thrombolytic enzyme properties of cattail pollen (Hyemin Park et al., 2009, CNU Journal of agricultural science v.36 no.1, pp. 111-122), antithrombosis of cattails , Hemostatic inhibitory effect (Zhao, G, 2011, Chinese journal of biochemical pharmaceutics 32: 222-224) is known, and it has been reported that the anticoagulant substance of caterpillar pollen is a high molecular polysaccharide (Gibbs, A et al., 1983, Thrombosis research 32 : 97-108). In addition, it has been reported that the extract of silsosangami-tang containing a pollen pollen inhibits platelet aggregation and fibrin aggregation (Park WH et al., 2004, Phytother Res. 18, 224-229). Therefore, it has been confirmed that caterpillar pollen exhibits antithrombotic activity due to thrombolytic activity, anticoagulant activity, and antiplatelet activity.

However, in the case of Songhwabun, the opposite activity of antithrombotic activity and hemostatic promoting activity is known. In studies related to antithrombotic activity of pine pollen, the lipid of pine pollen acts on platelets to promote aggregation reactions, or PAF (Platelet Activating Factor), a type of phospholipid that induces physiological activity. It has been reported to inhibit platelet aggregation and finally inhibit 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 pine pollen powder promotes platelet aggregation at high concentrations and mediates different reactions depending on the concentration, and the lipid component of pine pollen powder exhibits antithrombotic activity or opposite thrombus formation promoting activity depending on the treatment concentration. . However, until now, the anticoagulant and platelet aggregation inhibitory activity by water-soluble substances in sequential fractions of the extract of pine tree powder has not been known. In fact, most (~85%) of the pine pollen extract is a fat-soluble component, and the water-soluble water residue accounts for less than 1.9 g in 100 grams of pine pollen, so few studies have been conducted on the water-soluble ingredients of pine pollen.

As a patent related to blood clots in pollen, Korean Patent No. 10-0913370 discloses an antithrombotic composition targeting a thrombolytic enzyme, and WO-4009575 uses antithrombosis of pollen pollen [An extract of a typhae pollen and its manufacture and use], U.S. Patent Publication No. US-0018261 discloses an effect of improving blood circulation of the cattail pollen in [Method and use of extract of a member of Typhaceae's family].

In addition, as a patent related to pine pollen, [Song pollen extract is effective in Korean Patent Registration No. 10-1237215] [Pharmaceutical composition for preventing or treating enteritis] consisting of pine pollen, acorn pollen, honey, and propolis. Composition for enhancing congenital immunity and antiviral containing as an ingredient], Registration Patent 10-0438855 [Oral preparation for acne treatment containing Songhwa powder extract], Registration Patent 10-0407128 [External preparation for skin containing Songhwa powder or Songhwa powder extract ] Is known, and as a food-related patent, Patent Registration No. 10-1596213 [Method of manufacturing seasoned laver containing songhwa powder and seasoned laver produced thereby], registered patent No. 10-0838716 [addition of songhwa powder] The method of promoting the growth of Hwangguk bacteria or Naphthabacteria present in soybean paste], Registration Patent 10-0612778 [Nutrition Sundae containing Songhwa Powder], Registration Patent 10-0367425 [Healthy food using Songhwa Powder and its manufacturing method], Registered Patent 10-0637962 [Songhwa Sundubu Haejangguk and its manufacturing method] is disclosed. In addition, Korean Patent Publication No. 10-2013-0016001 Method for producing Songhwa Salt, 10-2017-0099071 Method for producing Songhwa Salt from which allergy-inducing properties are prevented, and Songhwa Salt produced thereby, 10-2000-0000855 Method for producing Songhwaju, 10-2001 -0048853 A toothpaste composition added with Songhwa Salt, 10-2010-0052302 A method for preparing soybean paste containing pineapple powder, and a method for producing soybean paste prepared by this method are disclosed.

However, until now, there are no studies or patent literatures on the antithrombotic activity of the pine pollen extract and its organic solvent fraction.

KR 10-0913370 B

Siafaka-Kapadai, A, Demopoulos, C A, Andrikopoulos, N K. 1986. Biochemistry international 12, 33-41

The present invention was conceived 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 the prevention or treatment of thrombosis and a health functional food containing a pine flower extract as an active ingredient. Is to do.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing the pine tree (Pollen of Pine tree) extract as an active ingredient.

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

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis containing the pine flower extract as an active ingredient.

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

Songhwamin extract as an active ingredient of a pharmaceutical composition for preventing or treating thrombosis and a health functional food of the present invention is due to the inhibitory effect of platelet aggregation, which plays a role in initiating thrombus formation along with inhibition of blood clotting-related enzymes and blood clotting factors. At the same time, it exhibits strong antithrombotic activity, does not show any hemolytic activity against human red blood cells, has excellent thermal stability, and shows a loss of blood coagulation factor inhibitory effect and blood clot formation-related enzyme inhibitory effect even in acidic conditions of pH 2 and plasma. Therefore, it is expected that it can 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, etc. It is a very useful invention in the pharmaceutical industry and food industry because it has an excellent effect that can be prepared with.

1 is a photographic view of Songhwabun.
Fig. 2 is a photographic view of pine flower powder observed under a microscope (200 times).
Figure 3 shows the human platelet aggregation inhibitory activity of the ethanol extract and its sequential organic solvent fractions: 1: solvent control (DMSO), 2: aspirin (0.25mg/ml), 3: aspirin (0.125mg/ml), 4: pine pollen ethanol extract (0.25 mg/ml), 5: hexene fraction of pine pollen ethanol extract (0.25 mg/ml), 6: ethylacetate fraction of pine pollen ethanol extract (0.25 mg/ml), 7: butanol of pine pollen ethanol extract Fraction (0.25 mg/ml), 8: Water residue (0.25 mg/ml) after organic solvent fractionation of the ethanol extract of pine pollen powder.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared pine pollen extract by a certain method in order to test the antithrombotic efficacy of pine pollen powder, and recovered the ethyl acetate fraction, butanol fraction and the water residue after the organic solvent fraction as an antithrombotic active ingredient, By confirming that the extracts and fractions do not exhibit any hemolytic activity against human red blood cells, but have excellent thermal stability and acid stability, the extracts and fractions are used as pharmaceutical compositions and health functional foods for the prevention or treatment/improvement of thrombosis. I tried to use it.

Specifically, the present inventors to develop a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis by using pine pollen, which is known to be effective in various diseases such as vascular and circulatory system, digestive system, metabolic system, aging, etc. , Hot water and ethanol extracts were prepared from pine pollen, and their antithrombotic activity was directly inhibited in human plasma and human thrombin (Thrombin Time), Prothrombin Inhibition (Prothrombin Time), active part thromboplastin time (activated) Partial Thromboplastin Time: aPTT) and platelet aggregation inhibitory activity were evaluated. As a result, it was confirmed that hot water extract had excellent platelet aggregation inhibitory activity and ethanol extract had good anticoagulant activity. Thereafter, the ethyl acetate fraction obtained by sequential organic solvent fractionation from the ethanol extract showed strong anticoagulant activity, and in the butanol fraction and the water residue after the organic solvent fraction, it was confirmed that the platelet agglutination inhibitory activity is much stronger than that of the previously known oil-soluble component of pineapple branch. In addition, it was confirmed that the ethanol extract showed strong human red blood cell hemolytic activity, but the butanol fraction and the water residue after the organic solvent fraction did not show any hemolytic activity against human red blood cells.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis, containing a pine tree extract as an active ingredient.

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

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis containing the pine tree (Pollen of Pine tree) extract as an active ingredient.

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

Hereinafter, the method for preparing the pine pollen extract and the active fractions of the present invention, and efficacy experiments will be described in more detail.

The present invention comprises the steps of preparing hot water and ethanol extract from pine pollen; Preparing sequential organic solvent fractions of hexene, ethyl acetate, and butanol from the ethanol extract of pine pollen and preparing a water residue obtained thereafter; Evaluating antithrombotic activity of the extracts and fractions; And a step of examining the stability of the ethyl acetate fraction, the butanol fraction, and the water residue after the organic solvent fraction.

The "songhwabun extract" included in the composition of the present invention may be obtained by extracting the pineapple powder with an organic solvent and filtering the extract using a filter net of 0.06mm 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 hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc. Can be used, hot water, or 95% ethanol extraction is most preferred.

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

In the present invention, as a result of measuring thrombin time, prothrombin time, and AP time at a concentration of 5 mg/ml of pine pollen extract, it showed weaker anticoagulant activity than aspirin (1.5 mg/ml) known as an antithrombotic agent, but ethanol extract It showed superior anticoagulant activity than this hot water extract. In particular, the ethanol extract of pine pollen powder exhibited anticoagulant activity similar to that of aspirin (1.5mg/ml) at a concentration of 6mg/ml. However, as a result of confirming the inhibition of platelet aggregation using the pine flower extract at a concentration of 0.25 mg/ml, the hot water extract showed an aggregation rate of 23.8%, showing almost similar platelet aggregation inhibitory activity to that of aspirin, whereas the ethanol extract showed an aggregation rate of 130.8%. And platelet aggregation promoting activity. However, in the case of the hot-water extract, the content of total sugar and reducing sugar was too high, so further purification was difficult, so the ethanol extract was sequentially fractionated with an organic solvent, and the antithrombotic activity of each fraction was evaluated.

As a result, strong inhibition of blood coagulation factor was observed in the hexene fraction and ethyl acetate fraction of the ethanol extract of pine pollen powder.Especially, the ethyl acetate fraction showed a 15-fold longer AP time compared to the non-added group at the concentration of 7 mg/ml. Inhibition of endogenous thrombus formation was confirmed. In addition, it was found that the butanol fraction and water residue also showed a concentration-dependent anticoagulant activity. Platelet aggregation inhibitory activity was confirmed in the butanol fraction and water extract (0.25mg/ml), a potent human platelet aggregation inhibition corresponding to aspirin. These results indicate that the ethylacetate fraction of the ethanol extract of pine pollen powder contributes to the inhibition of endogenous thrombus formation with its anticoagulant activity, and the butanol fraction and water residue have a very strong anti-platelet aggregation inhibitory activity, which has a high risk of side effects. It suggests that anticoagulants such as aspirin and antiplatelet drugs can be substituted.

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

The active ingredient of the present invention can be used for prevention or treatment of various diseases related to thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, motor abnormalities, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis. , Deep vein thrombosis, lower extremity swelling, pain, and acute peripheral arterial atresia. As venous thrombosis, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral sinus thrombosis, and central retinal vein occlusion.

Pharmaceutical compositions containing the active ingredients of the present invention are oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method for each purpose of use, and injections of sterile injectable solutions It may be formulated and used in various forms such as, and may be administered orally or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further include a carrier, excipient, or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, 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 contain a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

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

As a preferred embodiment, as a liquid formulation for oral use, a suspension, a liquid formulation, an emulsion, a syrup, and the like may be exemplified, and various excipients other than water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweetening agents, Fragrances, preservatives, etc. may be included.

As a preferred embodiment, the preparation for parenteral administration may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized agent, a suppository, 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 ethyloleate. Injectables may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to 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 disease, severity, drug activity, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

As a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kilogram of body weight daily Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the dosage amount is not limited by any method.

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

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, 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 an active ingredient to target cells.

In the present invention, the "object" is not particularly limited, but 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 variously used for 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, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules or beverages. .

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

The health functional food of the present invention may contain the compound as an essential component in the indicated ratio as an additional component, as well as food supplementary additives acceptable for food, such as natural carbohydrates and various flavoring agents.

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

Taumatin as the flavoring agent; Natural flavoring agents such as stevia, such as rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame, may 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 a variety of nutrients, vitamins, minerals, synthetic flavors, and flavoring agents such as natural flavors, colorants and thickeners, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juices, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion 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 one preferred specific example of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

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

Songhwa powder harvested in Andong, Gyeongbuk in 2018 was purchased and used to prepare hot water and ethanol extracts. Specifically, the ethanol extract of Songhwabun was prepared by adding 10 times the amount of ethanol to the Songhwabun sample, extracting twice at room temperature, collecting the extract, filtering, and concentrating under reduced pressure to obtain a powder. After sterilized distilled water was added and extracted twice at 100° C. for 1 hour, it was filtered and concentrated in the same manner as above.

The pine pollen powder used in the experiment was a yellow granule, showing a lightness of 64.27, a redness of 1.55, and a yellowness of 25.49, pH 6.0, brix 2.0, and acidity 0.1. The moisture content was 7.6%, which was in a very dry state, and 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), it was confirmed that the pine flower powder is a small elliptical spherical shape, and has a structure that can be easily scattered because the wings with oval net patterns are attached to both sides (Fig. 2).

[Table 1] Analysis of Physicochemical Characteristics and Color Difference of Songhwa Powder

As a result of nutrient analysis, crude protein 17.4%, crude fat 8.45%, crude carbohydrate 63.4%, 400kcal/100g, higher than general pollen by 100Kcal/100g, and ash content also showed 3.2%, which contained a large amount of various minerals. (Table 2).

[Table 2] Analysis of nutritional composition of Songhwa powder

The content of total polyphenols, total flavonoids, total sugars and reducing sugars was measured by component analysis of pine pollen extract. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of a saturated solution of Na ₂ CO _{3 were} added to 400 μl of the extracted sample solution, and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and then 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample solution, 40 μl of 1 N NaOH was added, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. Rutin was used as a standard reagent. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 3] Analysis of Extraction Efficiency and Useful Components of Ethanol and Hot Water Extract of Songhwa Powder

As shown in Table 3, the ethanol extraction efficiency of pine pollen powder was 6.3%, which was lower than the hot water extraction efficiency (12.1%), but showed a lower extraction efficiency of about 20% compared to the conventional chungmae flower pots (darae pots, acorn pots, etc.). . The total polyphenol content of the ethanol extract of pine pollen powder was 1.77 times higher than that of the hot water extract, and the total flavonoid content was also 2.9 times higher in the ethanol extract than the hot water extract. The content of total sugar and reducing sugar was 4.8 times and 2.7 times higher in hot water extract than ethanol extract, respectively, and it was found that most of hot water extract was composed of sugar.

Example 2: Evaluation of blood coagulation inhibitory activity of pine flower extract

The blood coagulation 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 coagulation inhibitory activity of pine flower 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 AP time were measured. Plasma was used as a commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and the thrombin time, prothrombin time, and APT measurement methods were performed as follows.

Thrombin Time

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

Prothrombin time

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

aPTT(activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to a tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added, and again at 37° C. Incubated for 3 minutes. Thereafter, after 50 μl CaCl ₂ (35 mM) was added, the time until plasma coagulation was measured. DMSO was used instead of the sample as a solvent control, and in this case, the coagulation time was 55.1 seconds. The result of aPTT was expressed as the average value of the experiment repeated three 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] Inhibitory Activities of Hot Water Extract and Ethanol Extract from Songhwabun

As a result, hot water and ethanol extract showed weak blood coagulation inhibitory activity at a concentration of 5 mg/ml, and ethanol extract showed aPTT prolongation due to blood coagulation factor inhibition. The extract exhibited concentration-dependent anticoagulant inhibition, and in particular, the ethanol extract exhibited better activity than aspirin (1.5mg/ml) at a concentration of 7mg/ml.

Example 3: Platelet Aggregation Inhibitory Activity of Songhwabun Extract

The hot water extract and ethanol extract of Example 1 were evaluated for inhibiting human platelet aggregation and the results are shown in Table 5. Platelets are disk-shaped small cells that circulate in blood vessels with various blood cells. Instead of having a nucleus, platelets have cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentrations, and aggregation when damage to the inner wall of blood vessels occurs. It is an important cell that initiates thrombus generation by secreting factors and forming a primary hemostatic plug by binding to collagen exposed due to damage of endothelial cells. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus formation. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were concentrated human platelets, which were washed once with a washing buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 1mM EDTA, pH 6.5). Thereafter, re-suspended 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), and then at 3,000 rpm for 10 minutes After centrifugation, it was resuspended in the suspending buffer again, 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 Hot Water Extract and Ethanol Extract

As shown in Table 5, first, aspirin exhibited 26.1% aggregation at 0.25mg/ml concentration and 45.4% aggregation at 0.125mg/ml concentration, confirming strong platelet aggregation inhibitory activity in a concentration-dependent manner. The basis for being used as a blood clot was found. On the other hand, the hot water extract of Songhwabun showed a degree of aggregation of 28.3% at 0.25mg/ml, showing excellent anti-aggregation activity, and the ethanol extract of Songhwabun promoted human platelet aggregation at a concentration of 0.25mg/ml, with a degree of aggregation of 130.6% compared to the non-added group Is shown. Therefore, it was determined that songhwabun contains various antithrombotic components and thrombus formation promoting components at the same time.

Example 4: Preparation of Sequential Organic Solvent Fractions of Ethanol Extract of Songhwabun and Analysis of Their Useful Components

The 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. Their fractionation efficiency and component analysis results are shown in Table 6.

[Table 6] Analysis of Yield and Useful Components of Organic Solvent Fractions of Ethanol Extract of Songhwa Powder

As shown in Table 6, the most part of the ethanol extract of pine pollen was transferred to the hexene fraction (64.7%), and the water residue accounted for 1.9% of the extract. This was in contrast to the conventional butanol fraction (50-65%) occupying the largest amount of the existing stalk and acorn pollen, and the water residues after fractionation accounted for 25-40% of the extract. This means that most of the pine pollen extract is a fat-soluble component that is soluble in oil, and contains almost no water-soluble substances. In fact, 0.12 g of water residue, 1.06 g of ethyl acetate fraction and butanol fraction, and 4.1 g of hexene fraction were recovered per 100 g of pine pollen. On the other hand, as a result of analysis of total polyphenol and total flavonoid content, the highest polyphenol content (62.3mg/g) and flavonoid content (33.3mg/g) in the ethyl acetate fraction were shown. This was 3.5 times higher polyphenol content and 8.3 times higher flavonoid content compared to the water residue. On the other hand, it could be inferred that the hexene fraction contains a very small amount of polyphenols and sugars, and most of them consist of lipids (oils).

Example 5: Anticoagulant activity of sequential organic solvent fractions from ethanol extract of pine pollen powder

The anticoagulant activity of the sequential organic solvent fractions obtained from the ethanol extract of pine pollen powder 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 Fractions from Ethanol Extract of Songhwabun

As a result, the ethanol extract of pine pollen powder exhibited better activity than aspirin (1.5mg/ml) at a concentration of 6-7mg/ml. Among its fractions, the hexene fraction and ethylacetate fraction prolonged AP time by inhibiting blood coagulation factors. Was confirmed. In particular, at a concentration of 7mg/ml of the ethyl acetate fraction, the AP time was extended by more than 15 times compared to the non-added group, indicating that strong endogenous thrombus formation inhibition was possible. In addition, concentration-dependent thrombin and prothrombin inhibition were confirmed in all fractions and water residues, and the best inhibitory activity was confirmed in ethyl acetate fraction. Therefore, it was confirmed that the anticoagulant activity was recognized in both the sequential organic solvent fraction and the water residue of the ethanol extract of pine pollen powder, and in particular, the ethyl acetate fraction excellently inhibited blood coagulation factors that contribute to the generation of endogenous thrombi.

Example 6: Human Platelet Aggregation Inhibitory Activity of Sequential Organic Solvent Fractions from Ethanol Extract of Songhwa Min.

Human platelet aggregation inhibitory activity of the sequential organic solvent fractions obtained from the ethanol extract of pine pollen powder prepared in Example 4 was evaluated in the same manner as in Example 3, and the results are shown in Table 8 and FIG.

[Table 8] Platelet Aggregation Inhibitory Activity of Sequential Organic Solvent Fractions from Ethanol Extract of Songhwa Min.

As shown in Table 8, the ethanol extract of Songhwamin promoted platelet aggregation, but the hexene fraction, butanol fraction, and water residue strongly inhibited human platelet aggregation.In particular, the butanol fraction and water residue were at 0.25mg/ml concentration. It exhibited an anti-aggregation activity similar to that of aspirin at the same concentration. In particular, as shown in FIG. 3, the hexene fraction showed strong initial platelet aggregation, but thereafter showed a form in which aggregation decreased. This is judged to be related to the fact that, as in the previous report (Siafaka-Kapadai, A, et al., 1986. Biochemistry international 12, 33-41), the effect of promoting platelet aggregation and inhibiting aggregation was exhibited according to the treatment concentration of the pineal powder lipid component. Therefore, it was judged that the lipid component of pine flower powder would be difficult to practically use as an antithrombotic agent. Therefore, it is believed that the butanol fraction and water residues of pine pollen extract can complement and replace existing antithrombotic agents such as aspirin, which are limited in use due to hemorrhagic side effects, gastrointestinal disorders, and hypersensitivity reactions.

Example 7: Human Red Blood Cell Hemolytic Activity of Ethanol Extract and Fractions thereof

Songhwa powder is a food that has been registered as a food ingredient and has secured safety. In order to evaluate the possibility of acute toxicity of the ethanol extract and its fractions, the hemolytic activity of human red blood cells was evaluated, and the results are shown in Table 9. At this time, the hemolytic activity was evaluated according to the previous report (Ho-Yong Son, 2014 ㆍKorean J. Microbiol. Biotechnol. 42: 285-292), and simply 100 μl of human red blood cells washed three times with PBS was placed in a 96-well microplate. Then, 100 μl of sample solution of various concentrations was added and reacted for 30 minutes at 37°C. After that, 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 for 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] Hemolytic activity of human red blood cells of ethanol extract and fractions thereof

First, it was confirmed that DMSO and water used as control cells had no hemolytic activity, and triton X-100 hemolytic 100% red blood cells at a concentration of 1 mg/ml. In addition, it was confirmed that amphotericin B, which is used as an anticancer agent and antifungal agent, hemolyzes 50% of red blood cells at a concentration of 0.00625mg/ml. On the other hand, the ethanol extract and the hexene fraction of the present invention showed strong red blood cell hemolytic activity, and each HC ₅₀ (concentration showing 50% red blood cell hemolytic activity) was calculated as 0.24 and 0.097 mg/ml. However, it was confirmed that the butanol fraction and water residue did not show any red blood cell hemolysis up to a concentration of 1 mg/ml, and thus did not have acute toxicity and red blood cell hemolytic activity. The ethyl acetate fraction showed 48.6% hemolysis at a concentration of 1 mg/ml, and no serious toxicity was observed.

Example 8: Plasma, acid and thermal stability evaluation of the ethanol extract and active fractions of pine pollen powder

Plasma stability, thermal stability, and acid stability against antithrombotic activity were confirmed for the ethanol extract and active fractions thereof obtained in Example 4 above. The extract and the active fraction were heat treated at 100° C. for 1 hour, treated at pH 2 (0.01M HCl) for 1 hour, and treated in plasma for 1 hour, and did not show a decrease in blood coagulation inhibition and platelet aggregation inhibitory activity. Therefore, it was confirmed that the pine pollen extract and its active fractions contained an antithrombotic active substance having acid resistance and heat resistance. As a result of the above, it was confirmed that the ethyl acetate fraction of the ethanol extract of Songhwamin contributes to the inhibition of endogenous thrombus formation through strong anticoagulant activity, and the butanol fraction and water residue contribute to the inhibition of thrombus formation through platelet aggregation inhibition.

Claims (4)

An anticoagulant containing an ethanol extract of Pollen of Pine tree as an active ingredient. The anticoagulant (anticoagulant) according to claim 1, wherein the ethanol extract of pine pollen powder is an ethyl acetate fraction of the ethanol extract of pine pollen powder. delete delete

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