KR102277057B1 - Pharmaceutical composition comprising the extraction of cattail 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 for preventing or treating thrombosis and a health functional food containing an extract of Pollen of Typha augustifolia L. as an active ingredient, and more particularly, to an ethanol extract of Pollen of Typha augustifolia L. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation, and a health functional food containing the ethyl acetate fraction of the active ingredient. As an active ingredient of the pharmaceutical composition for the prevention or treatment of thrombosis of the present invention, and as an active ingredient of a health functional food, the extract of dandelion pollen is effective in inhibiting platelet aggregation, which plays a role in initiating thrombus formation along with inhibition of thrombus formation-related enzymes and blood clotting factors At the same time, it exhibits strong antithrombotic activity and no hemolytic activity against human red blood cells, excellent thermal stability, and loss of blood coagulation factor inhibitory effect and thrombus formation-related enzyme inhibitory effect even in an acidic condition of pH 2 and plasma. Since it does not appear, 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 extracts, powders, pills, tablets, etc. It is a very useful invention in the pharmaceutical industry and the food industry because it has an excellent effect that it can be formulated in a form.

Description

A pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing extract of dandelion pollen as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing an extract of Pollen of Typha augustifolia L. as an active ingredient, and more particularly, to an ethanol extract of Pollen of Typha augustifolia L. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and inhibition of platelet aggregation, and a health functional food using the ethyl acetate fraction of

As a component of the human body, blood has a variety of important functions, such as transporting oxygen, nutrients, and waste products and buffering, maintaining body temperature, osmotic pressure control and ion balance maintenance, water constant maintenance, fluid control, blood pressure maintenance and control, and body defense. have. Normal blood circulation facilitates blood circulation as the blood coagulation and thrombolytic systems in the body are controlled complementary to each other, and the mechanism of the blood coagulation system among them is the adhesion and aggregation of platelets to the blood vessel wall to form a platelet thrombus. , it has been reported that the blood coagulation system is activated to form a fibrin clot centered on platelet agglomerates.

Fibrin thrombus formation is caused by the activation of thrombin, which is involved in fibrin coagulation, through a multi-step reaction of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen. The fibrin monomers are polymerized by calcium, and are absorbed into platelets and endothelial cells. It binds and forms a fibrin polymer cross-linked by factor XIII, forming a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation, such as activating platelets, factor V, and factor VII to promote a blood coagulation reaction. Therefore, the thrombin activity inhibitor can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood clotting.

In the endogenous thrombus formation pathway, it is known that the activation of prothrombin following the sequential activation of factors XII, XI, IX, and X ultimately activates thrombin, so specific inhibition of blood coagulation factors is also an important development target for thrombotic disease therapeutics. is becoming Until now, various anticoagulants such as heparin, coumarin, aspirin, urokinase, etc., antiplatelet agents, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases. As such, its use is limited.

On the other hand, Typha augustifolia L. is a perennial monocotyledonous plant of the Cataceae family, which grows in wetlands such as ponds, rivers, rivers, and swamps and can be seen in . , the leaves are linear, narrow, long, and green. In Korea, it is mainly used for ornamental purposes and flower arranging, and pollen (pollen) is used for hemostasis, pain relief, and diuresis in the private sector. In oriental medicine, in particular, pollen of perilla is called pohwang (蒲黃), and because it has a sweet taste and is not toxic, it is used as a hemostatic agent, uterine bleeding, hematemesis, hemorrhagic haemorrhage, prolapse, anti-inflammatory diuretic, hemorrhoids, hypothyroidism, menstrual irregularity, cystitis has been used as medicine.

Pollen is a reproductive organ of vascular plants that make seeds. It is a structure surrounded by a strong shell to protect the male gamete, and is mainly carried by wind or insects to reach the female gamete, fertilize, and form a seed. There are two types of pollen: insect-mediated and wind-mediated pollen. The pollen is divided into single plant pollen such as Actinidia and acorn pollen, as well as miscellaneous pollen collections of various plant pollens. You can pick up a potted plant.

Usually pollen contains 20~25% of crude protein, 7~15% of crude lipid, 2~5% of crude flour, 20~50% of crude carbohydrate, and 10~15% of water, so it is nutritionally excellent and contains various physiologically active substances. It has been used as a nutritional supplement and traditional medicine. However, pollen is composed of an extine and an intime composed of a thick layer called 'sporopollenin', so it is not digested or decomposed by itself, so there is a problem that the bioavailability is low at 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 spoiling. In addition, toxic substances (1,2-dehydro pyrollizidine alkaloids) suspected of hepatotoxicity, lung toxicity and carcinogenicity have been identified in borage pollen (Boppre, M et al., J. Agric. Food Chem. 53, 594-). 600).

Therefore, research on the existing pollen is mainly focused on the development of extraction conditions and pretreatment methods to improve digestion and absorption by decomposing the nutritional properties of pollen and the strong outer shell. Recently, research on useful physiological activity is being conducted, and it is known that it is effective in various diseases such as blood vessels and circulation, digestive system, metabolic system, aging, etc., antibacterial and antioxidant efficacy, immune enhancing effect, prostate enlargement 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 In-pyo et al., 2016, Journal of Apiculture 31, 219-225; Hong In-pyo, 2014, Journal of Apiculture 29, 137-142; Park Yong-gi et al., 2015. Journal of Apiculture 30, 299-306; Kim Seok-jung et al., 2005 , Journal of the Korean Society for Food Science 37, 833-837).

On the other hand, studies on thrombosis of pollen have been reported very limitedly only in pine pollen and pine pollen, and in particular, in the case of pine pollen, opposite activities of antithrombotic and hemostasis promotion are known at the same time. well known (Ohkura et al., 2011. Blood Coagul Fibrinolysis. 22:631-636; Yan X., 2017, J Nanobiotechnology. 15: 60 doi: 10.1186/s12951-017-0296-z.)

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). . First, as a study related to the antithrombotic activity of cattail 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), Antithrombotic and hemostatic inhibitory effects (Zhao, G, 2011, Chinese journal of biochemical pharmaceutics 32: 222-224) are known, and it has been reported that the anticoagulant of pollen pollen is a high-molecular polysaccharide (Gibbs, A et al., 1983, Thrombosis). research 32: 97-108). In addition, it has been reported that 7 kinds of herbal medicine ventral extract (Silsosangami-tang) including perilla pollen, Oryeongji, peony, cheongung, doin, safflower and bongchul inhibits platelet aggregation and fibrin aggregation (Park WH et al., 2004, Phytother Res. 18, 224-229).

In addition, in studies related to the antithrombotic activity of pine pollen obtained from pine, the lipid component of pine pollen acts on platelets to promote aggregation reaction, or inhibits PAF (Platelet Activating Factor), a type of phospholipid that induces physiological activity. Thus, it has been reported that ultimately 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 such pine pollen mediates different reactions depending on the concentration by promoting platelet aggregation at high concentrations. Therefore, in the case of pine pollen, depending on [ingredient composition and treatment concentration], antithrombotic activity or the opposite thrombus formation promoting activity is exhibited. However, there are no reports of low-molecular active substances in ethanol extracts of pollen pollen.

In addition, as patents related to thrombus in pollen, Korean Patent Registration No. 10-0913370 [Antithrombotic Composition Containing Pollen Pollen Extract] targeting a thrombolytic enzyme is known, and WO-4009575 , Antithrombosis of Pollen Pollen Use [An extract of a typhae pollen and its manufacture and use], U.S. Patent Publication No. US-0018261 discloses the blood circulation improving effect of pollen pollen [Method and use of extract of a member of Typhaceae's family]. However, until now, there has been no known patent for antithrombotic activity of a pollen extract and a fraction containing a fat-soluble low molecular weight thereof as an active ingredient.

US 10-0913370 B

Hyemin Park et al., 2009, CNU Journal of agricultural science v.36 no.1 ,pp. 111-122 Zhao, G, 2011, Chinese journal of biochemical pharmaceutics 32: 222-224 Gibbs, A et al., 1983, Thrombosis research 32: 97-108

The present invention has been devised to solve the problems of the prior art as described above, and an object 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 an extract of pollen pollen of the present invention as an active ingredient. would like to

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing an extract of pollen of Typha augustifolia L. as an active ingredient.

Preferably, the pollen extract is ethanol extract of pollen pollen.

It is preferable that the ethanolic extract of pollen pollen from Pollen is an ethyl acetate fraction of the ethanol extract of Pollen Pollen.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing an extract of pollen of Typha augustifolia L. as an active ingredient.

Preferably, the pollen extract is ethanol extract of pollen pollen.

It is preferable that the ethanolic extract of pollen pollen from Pollen is an ethyl acetate fraction of the ethanol extract of Pollen Pollen.

As an active ingredient of the pharmaceutical composition for the prevention or treatment of thrombosis of the present invention, and as an active ingredient of a health functional food, the extract of pollen pollen from thrombosis is effective in inhibiting platelet aggregation, which plays a role in initiating thrombus formation along with inhibition of thrombus formation-related enzymes and blood clotting factors At the same time, it exhibits strong antithrombotic activity and no hemolytic activity against human red blood cells, excellent thermal stability, and loss of blood coagulation factor inhibitory effect and thrombus formation-related enzyme inhibitory effect even in an acidic condition 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 extracts, powders, pills, tablets, etc. It is a very useful invention in the pharmaceutical industry and food industry because it has an excellent effect that it can be formulated in a form.

1 is a photographic diagram of a potted plant.
Figure 2 is a microscopic (200 times) observation photograph of the pollen of Cattail.
Figure 3 shows the human platelet aggregation inhibitory activity of an ethanolic extract and sequential organic solvent fractions thereof: 1: solvent control (DMSO), 2: aspirin (0.25 mg/ml), 3: ethanolic extract of dandelion pollen (0.25 mg). /ml), 4: Hexene fraction (0.25 mg/ml) of ethanolic extract of Pollen plant asiatica, 5: Ethyl acetate fraction (0.25 mg/ml) of ethanol extract of Pollen fern pollen, 6: Butanol fraction (0.25 mg/ml) of ethanolic extract of Pollen Pollen. ml), 7: Water residue (0.25 mg/ml) after organic solvent fractionation of ethanol extract of pollen pollen.

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of pollen of the present invention, the inventors of the present invention prepared a pollen extract by a certain method, and an ethyl acetate fraction thereof was recovered as an antithrombotic active ingredient, and the ethyl acetate fraction was used for human red blood cells. By confirming that it has excellent thermal stability and acid stability without showing any hemolytic activity, the extracts and fractions were intended to be utilized as pharmaceutical compositions and health functional foods for preventing or treating/improving thrombosis.

Specifically, the present inventors in the folklore to develop pharmaceutical compositions and health functional foods for the prevention or treatment/improvement of thrombosis by using pollen pollen, which is known to be effective in various diseases such as blood vessels and circulatory system, digestive system, and metabolic system. , prepared ethanol extracts from pollen of sagebrush, and tested their antithrombotic activity against human plasma and human thrombin for direct thrombin inhibition (Thrombin Time), prothrombin inhibition (Prothrombin Time) and active partial thromboplastin time (activated partial time). Thromboplastin Time: aPTT) was evaluated to confirm that it had excellent anticoagulant activity. The hexene and ethyl acetate fractions obtained by sequential organic solvent fractionation of the ethanol extract showed strong blood coagulation factor inhibitory activity, and the ethyl acetate fraction of Pollen pollen extract showed strong anticoagulant activity. A strong inhibition of platelet aggregation was confirmed. In addition, it was confirmed that the ethyl acetate fraction did not show hemolytic activity against human red blood cells, confirming that it was practically usable.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing an extract of pollen of Typha augustifolia L. as an active ingredient.

Preferably, the pollen extract is ethanol extract of pollen pollen.

It is preferable that the ethanolic extract of pollen pollen from Pollen is an ethyl acetate fraction of the ethanol extract of Pollen Pollen.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing an extract of pollen of Typha augustifolia L. as an active ingredient.

Preferably, the pollen extract is ethanol extract of pollen pollen.

It is preferable that the ethanolic extract of pollen pollen from Pollen is an ethyl acetate fraction of the ethanol extract of Pollen Pollen.

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

The present invention comprises the steps of preparing an extract from pollen of Cattail; a step of preparing sequential organic solvent fractions of hexene, ethyl acetate, and butanol from the pollen extract, and preparing a water residue obtained thereafter; It includes a step of evaluating the antithrombotic activity of the extract and the fraction and a step of examining the stability of the active substance of the ethyl acetate fraction.

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

The organic solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous 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, and hot water or 95% ethanol extraction is most preferred.

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

In the present invention, as a result of measuring thrombin time, prothrombin time, and AP time by using a pollen extract of safflower pollen at a concentration of 5 mg/ml, the hot water extract had weaker anticoagulant activity than aspirin (1.5 mg/ml), which is known as an antithrombotic agent. However, the ethanol extract showed superior prothrombin inhibitory activity and blood coagulation factor inhibitory activity than aspirin.

However, as a result of confirming inhibition of platelet aggregation at a concentration of 0.25 mg/ml of coriander pollen extract, the hot-water extract exhibited 154.1% aggregation rate and 318.8% aggregation rate in the ethanol extract compared to the no-addition control, indicating strong platelet aggregation promotion. These results can be understood as the basis for using the existing cattail pollen as a powerful hemostatic agent in folk and oriental medicine. Thereafter, the organic solvent fractionation was sequentially performed on the ethanol extract, which was relatively easy to purify the active material due to the low total sugar and reducing sugar content, and had excellent anticoagulant activity. As a result, strong inhibition of blood coagulation was shown in the hexene fraction and the ethyl acetate fraction of the pollen ethanol extract, and in particular, in the ethyl acetate fraction, at a concentration of 5 mg/ml, thrombin time, prothrombin time, and AP time were respectively added to the non-additive group. It was confirmed that the thrombin time, prothrombin time, and AP time were extended by 7.73 times, 2.51 times, and 15 times or more, respectively, at the concentration of 6 mg/ml. In addition, as a result of evaluating the platelet aggregation inhibitory activity of the fraction, it was confirmed that the ethyl acetate fraction exhibited a stronger inhibition of human platelet aggregation at a concentration of 0.25 mg/ml than aspirin at the same concentration. These results indicate that the ethyl acetate fraction of the pollen extract of Cat. pollen has antithrombotic activity through very strong anticoagulant and platelet aggregation inhibitory activity, and that it can replace the existing anticoagulant and antiplatelet agents such as aspirin, which have high side effects. present.

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

The active ingredient of the present invention can be used for prevention or treatment of various diseases related to thrombosis. These diseases include, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, blurred vision, epileptic seizures, pulmonary thrombosis , deep vein thrombosis, lower extremity edema, pain and acute peripheral arterial occlusion, and the like. Examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral vein sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention can be prepared according to a conventional method for each purpose of use, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injection solutions. It can be formulated and used in various forms, such as oral administration, or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

The pharmaceutical composition 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 include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like.

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

As a preferred embodiment, the oral liquid formulation may be exemplified by a suspension, an internal solution, an emulsion, a syrup, etc., and various excipients, for example, a wetting agent, a sweetening agent, in addition to water and liquid paraffin, which are commonly used simple diluents, Perfumes, preservatives, and the like may be included.

As a preferred embodiment, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents, suppositories, etc. can be exemplified as preparations for parenteral administration. 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 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" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, 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 may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum 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 age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg 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, disease severity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject through various routes. Any mode of administration can be envisaged, for example, by 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 administration route 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 a target cell.

In the present invention, "subject" is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. and preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in various ways, such as food and beverage effective for preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gum, tea, vitamin complexes, health supplements, and the like, and may be used in powder, granule, tablet, capsule or beverage form. .

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 a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

In addition to containing the above compound as an essential ingredient in the proportion indicated, the health functional food of the present invention may contain food pharmaceutically acceptable food supplement additives, such as natural carbohydrates and various flavoring agents, as additional ingredients.

Examples of the natural carbohydrate 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.

The flavoring agent includes: thaumatin; A natural flavoring agent such as stevia, such as rebaudioside A or glycyrrhizin, and a synthetic flavoring agent such as saccharin or 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 various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloids It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice for the production of fruit juice drinks, vegetable drinks, and the like. These components may be used independently or in combination. The ratio of these additives is generally selected in the range of 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 scope of the following examples.

[Example]

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

Potted plants harvested in Andong, Gyeongsangbuk-do in 2018 were purchased and used to prepare hot water and ethanol extracts. Specifically, 10 times of ethanol was added to the pollen pollen sample, extracted twice at room temperature, the extract was collected, filtered, and concentrated under reduced pressure to prepare a powder to prepare an ethanol extract, which was subjected to hexene and ethyl acetate. , organic solvent fractionation was sequentially with butanol, and finally the water residue was recovered. In the case of the hot water extract of dandelion pollen, 10 times of sterile distilled water was added to the pollen pollen sample, and the extraction was repeated twice for 1 hour at 100°C, followed by filtration and concentration in the same manner as above.

The pollen pollen used in the experiment was dark yellow fine granules, and had a brightness of 59.44, a redness of 0.35, and a yellowness of 30.49, and had a pH of 5.6, brix 20.0, and acidity of 0.51, indicating a slightly sour sweetness. The moisture content was 12.5%, which was a state in which moisture could be absorbed again when left at room temperature for a long time in a dried state (FIG. 1, Table 1). As a result of observation under a microscope (200 times), the pollen was similar to an acorn pollen with a spherical shape and a granular surface, and was differentiated from the pine pollen, which is a representative wind plum pollen, because it did not have a reticulated wing structure (FIG. 2).

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

As a result of nutrient analysis, 18.2% of crude protein, 1.0% of crude fat, and 64.1% of crude carbohydrate were excellent at 338 kcal/100g, and ash content was also 4.2%, which included a large amount of various minerals (Table 2).

[Table 2] Nutrient analysis of pollen pollen

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

[Table 3] Extraction efficiency and useful components analysis of ethanol and hot water extract

As shown in Table 3, the ethanol extraction efficiency of pollen pollen was 12.5%, which was lower than that of hot water extraction (29.8%). In addition, the total polyphenol content of the ethanol extract was lower at 57.4% than that of the hot water extract, and the total flavonoid content was similar in the hot water extract and the ethanol extract. In addition, the total sugar and reducing sugar contents were 2.7 times and 2.65 times higher in the hot water extract than in the ethanol extract, indicating that most of the hot water extract was composed of sugar.

Example 2: Evaluation of blood clotting inhibitory activity of pollen extract

The blood coagulation inhibitory activity of the ethanol extract and hot water extract of Example 1 was evaluated, and the results are shown in Table 4. In this case, the blood coagulation inhibitory activity of the 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 AP time were measured. For plasma, commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and thrombin time, prothrombin time and AP were performed as follows.

Thrombin Time

At 37°C, 50 μl of 0.5U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ , and 10 μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, and then 100 μl of plasma was collected. After the addition, 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. DMSO showed a coagulation time of 32.1 seconds. The thrombin inhibitory effect was expressed as an average of three or more repeated experiments.

prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solutions of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, 130 μl of PT reagent was added, and plasma was coagulated. The time until completion 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 clotting time was 18.1 seconds. The prothrombin inhibitory activity was expressed as a value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

Activated Partial Thromboplastin Time (aPTT)

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

[Table 4] Blood clotting inhibitory activity of ethanol extract and hot water extract

As a result, as shown in Table 4, the hot water extract showed weak blood coagulation inhibitory activity at the concentration of 5 mg/ml, whereas the ethanol extract showed better prothrombin than aspirin (1.5 mg/ml), which is used as an antithrombotic agent at the concentration of 5 mg/ml. and blood coagulation factor inhibitory activity (APT time).

Example 3: Platelet aggregation inhibitory activity of Pollen Pollen Extract

The human platelet aggregation inhibitory activity of the ethanol extract and hot water extract of Example 1 was evaluated, and the results are shown in Table 5. Platelets are disk-shaped small cells that circulate in blood vessels together with various blood cells. Instead of having a nucleus, they have cytoplasmic granules containing various substances related to vascular damage protection and platelet aggregation in high concentrations. It is an important cell that secretes factors and initiates thrombus formation by forming a primary hemostatic plug by binding to collagen exposed due to endothelial cell damage. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus formation to be. The platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were human concentrated 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). Then, 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 suspending buffer, and the platelet count was adjusted to be ^{4x10 9 /ml.} Then, 2.5 μl collagen was added to the 1 ml suspension, reacted 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 an aggregation of 42.3% at a concentration of 0.25 mg/ml, indicating an excellent inhibition of platelet aggregation, thereby confirming the rationale for its use as an antithrombotic agent in clinical practice. On the other hand, the hot water extract of dandelion pollen promotes 154.1% of human platelet aggregation at 0.25 mg/ml and the ethanol extract at a concentration of 0.25 mg/ml of 318.8%, and the pollen extract shows strong platelet aggregation activity, so it is used as a hemostatic agent in folk and oriental medicine. could understand the rationale for In addition, it was found that the ethanol extract of pollen pollen had anticoagulant activity and platelet aggregation promoting activity, and simultaneously had opposite activities of thrombus formation inhibition and thrombus formation promoting activity. In fact, pollen pollen is used in oriental medicine for the opposing efficacy of a hemostatic agent and removal of blood stasis.

Example 4: Preparation of Sequential Organic Solvent Fractions of Ethanol Extract of Pollen Pollen and Their Useful Component Analysis

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 fractions of ethanol extract of pollen pollen

As shown in Table 6, it was found that the largest portion of the pollen extracts of Cat. pollen was the hexene fraction (70.3%), and it was found that the ethanol extract of the pollen of Cat. pollen contained a significant amount of fat-soluble components, and the butanol fraction and the water residue were respectively the fraction of the extract. 12.8% and 12.1%. The ethyl acetate fraction exhibiting the lowest yield represented 9.5% of the pollen extract, 1.18 g per 100 g of cattail pollen could be recovered.

On the other hand, as a result of analysis of the total polyphenol and total flavonoid content, the ethyl acetate fraction showed the highest polyphenol content (89.0 mg/g) and flavonoid content (101.6 mg/g). This was 9.1 times higher polyphenol content and 5.95 times higher flavonoid content compared to the water residue with the lowest content. Therefore, it was expected that the ethyl acetate fraction of the pollen pollen extract would exhibit various physiological activities, and as a result of actual analysis, the fraction exhibited strong antioxidant activity and nitrite scavenging ability. Therefore, it is expected that the ethyl acetate fraction of the pollen pollen will additionally contribute to the improvement of blood circulation and antithrombotic activity by exhibiting excellent antioxidant and carcinogenic activity.

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

The anticoagulant activity of the sequential organic solvent fractions of the 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 fractions of Ethanol extract of pollen pollen

Among the fractions of the pollen extract, the butanol fraction and the water residue showed no anticoagulant activity, but the hexene fraction and the ethyl acetate fraction showed strong inhibition of thrombin and blood coagulation factors. In particular, it was confirmed that the ethyl acetate fraction exhibited strong anticoagulant activity by excellently inhibiting blood coagulation factors contributing to endogenous thrombus formation.

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

The human platelet aggregation inhibitory activity of the sequential organic solvent fractions of the 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 fractions of Ethanol extract of pollen pollen

As a result, human platelet aggregation was strongly observed only in the ethylacetate fraction of the ethanol extract of pollen pollen, and showed a platelet aggregation activity of 8% at a concentration of 0.25 mg/ml. This is a better inhibition of aggregation than aspirin at the same concentration, suggesting that the ethyl acetate fraction of pollen pollen can supplement and replace existing antithrombotic agents, such as aspirin, whose use is limited due to bleeding side effects, gastrointestinal disorders, and hypersensitivity reactions. have. The hexene fraction, which occupies the largest amount of pollen fractions, exhibited a degree of aggregation of 170% or more, which was expected to show a hemostatic effect by promoting platelet aggregation.

Example 7: Human Erythrocyte Hemolytic Activity of Cattail Pollen Extract and Fractions Thereof

Pollen pollen is registered as a food raw material, so safety is secured, and it is edible by itself, and is also used as an auxiliary material and additive for various food processing. Human red blood cell hemolytic activity was evaluated in order to evaluate the acute toxicity potential of Cat. pollen extract and its fractions, 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 100 μl of human red blood cells washed three times with PBS was simply added to a 96-well microplate. 100 μl of sample solutions of various concentrations were added, followed by reaction at 37° C. for 30 minutes. After that, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414 nm. measured. DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1 mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 9] Human erythrocyte hemolytic activity of Cat. pollen extract and fractions thereof

First, DMSO and water used as controls had no hemolytic activity, and it was confirmed that triton X-100 hemolyzed red blood cells 100% at a concentration of 1 mg/ml. In addition, in the case of amphotericin B, which is used as an anticancer and antifungal agent, it was confirmed that more than 50% of red blood cells were hemolyzed at a concentration of 0.025 mg/ml. Fractions other than the ethanol extract and hexene fraction of the pollen of the present invention did not show any red blood cell hemolysis at a concentration of 1 mg/ml, confirming that there was no acute toxicity and no red blood cell hemolytic activity. The hexene fraction showed a hemolytic activity of 73.9% at 1mg/ml, 67.8% at 0.5mg/ml, and 30.5% at 0.25mg/ml, so it was judged to have acute toxicity potential, and the ethanol extract was also 82.7 at 1mg/ml. %, 93.2% at 0.5mg/ml, and 41.6% at 0.25mg/ml, it was judged that there would be restrictions on practical use.

Example 8: Evaluation of Plasma, Acid and Thermal Stability of Ethyl Acetate Fraction of Ethanol Extract of Cattail Pollen Pollen

Plasma stability, thermal stability and acid stability for antithrombotic activity were confirmed for the ethyl acetate fraction obtained in Example 4. The active fraction of the pollen of dandelion did not show any decrease in blood coagulation inhibitory and platelet aggregation inhibitory activity even after 1 hour heat treatment at 100° C., 1 hour treatment at pH 2 (0.01 M HCl), and 1 hour treatment in plasma. Therefore, it was confirmed that the ethyl acetate fraction of pollen pollen contains antithrombotic active substances of various components having acid resistance and heat resistance.

As shown in the above results, it was suggested that the ethyl acetate fraction of the pollen extract of Cat. pollen exhibited excellent anticoagulant and platelet aggregation inhibitory activity, but did not have hemolytic activity against human red blood cells, suggesting that it could be used as a practical antithrombotic agent.

Claims (4)

A pharmaceutical composition for preventing or treating thrombosis, comprising an ethyl acetate fraction of Pollen of Typha augustifolia L. ethanol extract as an active ingredient. An anticoagulant containing the ethyl acetate fraction of Pollen of Typha augustifolia L. ethanol extract as an active ingredient. An antiplatelet agent comprising the ethyl acetate fraction of Pollen of Typha augustifolia L. ethanol extract as an active ingredient. A health functional food for preventing or improving thrombosis, comprising the ethyl acetate fraction of Pollen of Typha augustifolia L. ethanol extract.

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