KR20200038717A - Pharmaceutical composition comprising the extraction of flower apple as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating thrombosis, comprising an extract of Malus prunifoliaWild. Borkh as an effective component. More specifically, the extract of Malus prunifoliaWild. Borkh of the present invention, as an effective component of a pharmaceutical composition and a health functional food for preventing or treating thrombosis, exhibits potent antithrombotic activity via inhibition on enzymes involved in thrombus formation and blood coagulating factors, and inhibition of platelet aggregation, while exhibiting no lytic activity to human red blood cells; has excellent thermal stability; and does not show a loss of blood coagulation inhibition and platelet aggregation inhibition effects even in acidic conditions of pH 2 and blood plasma, and thus, through improving blood circulation, is expected to be usable as a pharmaceutical composition and a health functional food for preventing and treating/alleviating thrombosis, such as ischemic stroke and hemorrhagic stroke. Further, the effective component may be processed into various forms such as extract, powder, pills, and tablets, and prepared in a form that can be taken at any time, and thus is extremely useful in the drug and food industries.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE EXTRACTION OF FLOWER APPLE 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 for preventing or treating thrombosis and a health functional food containing a flower apple ( Malus prunifolia Wild. Borkh) extract as an active ingredient, and more specifically, an extract of a flower apple mature fruit It relates to a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis through strong blood clotting inhibition and platelet aggregation inhibition using as an active ingredient.

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

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

On the other hand, the flower apple is a deciduous broad-leaved tree belonging to the family Rosaceae (Malus), and its origin is Europe, Asia, and North America. In English, it is called Flower Apple, Plum-leaved Apple, Chines Apple, and Crab Apple. Flower apples grow very well even in barren land, and they are used as rootstock of apple trees, which are difficult to self-fertilize because they bloom a lot of flowers at once, and are also used as garden trees because of their colorful flowers.

The fruit of the flower apple is much smaller than ordinary apples, and is rich in fructose, glucose, tartaric acid, and vitamin C. It is said to be good for health maintenance, fatigue recovery, and constipation, but it is almost edible for food due to the unique sourness and low sensory properties of the fruit. It is not used and is mostly discarded due to lack of water power. In the private sector, it was also used as a softening agent to soften meat when cooking meat. Soaking it with a flower apple has the effect of healing fatigue, improving appetite, nervousness, insomnia, and constipation. In oriental medicine, dried fruits are used for stomach and indigestion, anorexia, gastric acid deficiency or hyperacidity, and are known to be used as medicines for diarrhea, menstrual pain, frostbite, dry stomach, low back pain, and intestinal bleeding.

Flower apples have no genetic barriers and are characterized by free cross-species or intra-species hybridization. Over 700 varieties have been developed worldwide. The flower color is white, light pink, pink, dark pink, crimson, etc. The flowering period is 8 ~ 12 days, blooming in April, and the fruit is known to be less than 2.5cm in diameter (Song Dong-Young. 2011, Konkuk University Master's Thesis) ). In addition, the flower apple has a strong cold resistance, so it is excellent in environmental adaptability, and has the characteristic of growing well in barren land. In the case of apple trees, the flowering time differs for each variety, and there is a difference in insect pests and cultivation methods, so the number of pollinated varieties is required. The most commonly grown flower apple varieties in Korea are Manchurian, Hopay, Adams, and Prosper Sprenger. , Floret, SKK-14, SKK-16 and others.

Normally, in the private sector, the fruits of the hawthorn (Chinese hawthorn Crataegus cuneata Sieb, Western hawthorn Crataegus oxyacantha L.) are similar to the flowering apples, and are often called chaotic, but this should be classified as wrong.

As for research related to flower apples, selection of flower apples suitable for hydration by apple varieties (Ingyu Kang et al., 2002. Horticultural Science Journal 20: 330-334; Son Kwang-min et al., 2013. Protected Horticulture and Plant Factory, 22: 116- 122), Research on the reason why it grows well in barren land (Huang L. et al., 2018, Plant Physiol Biochem. 127: 185-193; Tan Y et al., 2017, Plant Physiol Biochem. 115: 219-228) It has been reported that the anthocyanin pigment of the flower apple is excellent in light resistance and heat resistance, so that it can be easily used as a natural red pigment resource (Yonghwan Kim, 1999, Korean J. Food Nutr. 12: 85-90). However, to date, studies on useful components and useful physiological activities of flower apples are very limited.

Recently, it has been reported that the polyphenol and flavonoid components of the flower apple are more than twice that of ordinary apples, and are rich in tartaric acid and vitamin C (Song Dong-young. 2011, master's thesis of Konkuk University). Gallic acid, protocatechuic than ordinary apples It has been reported that antioxidants are higher than apples due to high phenolic acid content such as acid, chlorogenic acid, p-coumaric acid and ferulic acid, and high flavonoid content such as quercetin and myricetin (KM Maria John et al., 2014. Food Chemistry 163: 46-50). Also, recently, the flower apple Malus 'Red jade', Malus hupehensis (Pamp.) Rehd. And Malus prunifolia (Willd.) Borkh from three species, citric acid, p-coumaric acid, hyperoside, myricetin, naringenin, quercetin, kampferol, gentiopicroside, ursolic acid and 8-epiloganic acid, and their cholesterol lowering and hyperlipidemia prevention effect Has been reported (Chao Wen et al., 2018. J. Pharmaceutical Biomedical Analysis 150: 144-151). However, until now, the strong coagulation inhibitory and platelet aggregation inhibitory activity of the flower apple extract has not been known.

For patents related to flower apples, refer to Korean Patent Registration No. 10-1081059 [Flower mixture extract having antioxidant and whitening activity and method for extracting it and cosmetic composition containing the flower mixture extract] and Japanese patent JP-0137455 [ Production of nursery stock of pear tree] is disclosed, and the patent or research results for the active extract of non-toxic flower apples, while showing strong antithrombotic activity through strong blood clotting inhibition and platelet aggregation inhibition, There is no bar.

 Chao Wen et al., 2018.J. Pharmaceutical Biomedical Analysis 150: 144-151

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

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing the flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple extract is preferably a hot water or ethanol extract of a flower apple variety selected from the group consisting of Adams, Professor Sprinkler and Manchurian.

The flower apple extract is preferably an ethanol extract of Flore variety.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing the flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple extract is preferably a hot water or ethanol extract of a flower apple variety selected from the group consisting of Adams, Professor Sprinkler and Manchurian.

The flower apple extract is preferably an ethanol extract of Flore variety.

 The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the flower apple extract as an active ingredient of a health functional food exhibit strong antithrombotic activity through inhibition of platelet aggregation and inhibition of enzymes and blood clotting factors related to thrombus production, and at the same time, human Thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation because it does not show any hemolytic activity against red blood cells, has excellent thermal stability, and does not exhibit blood clotting and platelet aggregation inhibitory effects even in acidic conditions and plasma of pH 2. It is expected to be used as a pharmaceutical composition for prevention and treatment / improvement and health functional food, and the active ingredient is processed into various forms such as extract, powder, pill, tablet, etc. Effective, very useful inventors in the pharmaceutical and food industries A.

Figure 1 shows the flower apple varieties used in the Examples of the present invention, showing Adams, Hopey, Flore, Prosper Sprenger and Manchurian varieties, respectively, from the left.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, in order to test the anti-thrombotic efficacy against the flower apple, after harvesting the flower apple to remove the foreign matter, prepare a hot water and ethanol extract in a certain way, evaluate the activity of the flower exhibiting antithrombotic activity The apple extract was identified, and the active extract does not show any hemolytic activity against human red blood cells, but confirms that it has excellent thermal stability and acid stability, thereby preventing or treating / improving the flower apple extract with thrombosis. It was intended to be used as a composition and dietary supplement.

Specifically, the present inventors have developed various types of flower apples (Adams, Hopayia) to develop pharmaceutical compositions and health functional foods for prevention or treatment / improvement of thrombosis using flower apples that are used for various purposes in the private sector. , Flore, Prosercer Sprenger, Manchurian), respectively, to prepare a hot water extract and ethanol extract, respectively, their antithrombotic activity directly inhibited thrombin against human plasma and human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin) Time) and activated Partial Thromboplastin Time (apTTT) were evaluated. As a result, strong anticoagulant activity was confirmed in hot water extracts and ethanol extracts of the Adams, Prosercer, and Manchurian varieties among the flower apples. This antithrombotic activity was a strong antithrombotic activity that surpasses aspirin used as an antithrombotic agent in clinical practice. In addition, as a result of evaluating the ability of the hot water extract and the ethanol extract to inhibit human platelet aggregation, it was confirmed that the platelet aggregation inhibitory ability was excellent in the ethanol extracts of Adams, Flore, Prosercer Sprinkler, and Manchurian varieties. In addition, it was confirmed that all of the flower apple extract does not show hemolytic activity against human red blood cells.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing the flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple extract is preferably a hot water or ethanol extract of a flower apple variety selected from the group consisting of Adams, Professor Sprinkler and Manchurian.

The flower apple extract is preferably an ethanol extract of Flore variety.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing the flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple extract is preferably a hot water or ethanol extract of a flower apple variety selected from the group consisting of Adams, Professor Sprinkler and Manchurian.

The flower apple extract is preferably an ethanol extract of Flore variety.

Hereinafter, a method of manufacturing the flower apple extract of the present invention and an efficacy test will be described in more detail.

The present invention harvesting a variety of varieties of flower apples, removing foreign matter; Slicing a flower apple and preparing an extract; Evaluation step of anti-thrombotic activity of flower apple extract; And a stability investigation step of the active extract.

"Flower apple" of the present invention means a mature flower apple in which ovaries were produced more than 130 days after flowering. In the present invention, the flower apples of Adams, Hopay, Floret, Prosercer Sprenger, and Manchurian varieties were used.

"Flower apple extract" contained in the composition of the present invention comprises the steps of crushing a mature flower apple; Extracting the flower apple crushed material with an organic solvent; Filtering the extract using a filter network of 0.06 mm or less; And concentrating it under reduced pressure.

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

 Among the anticoagulant activity of the flower apple hot water extract of the present invention, thrombin and prothrombin inhibitory activity were Adams> Professor Sprinkler> Manchurian> Hopayi> Flore, and the blood coagulation factor inhibitory activity exhibited by Apity time was Adams> Manchurian> It was similar in the order of Professor Sprenger> Hopey> Flore. In addition, thrombin and prothrombin inhibitory activity among the anticoagulant activity of the flower apple ethanol extract was in the order of Adams> Prosper Sprenger> Manchurian> Hopei> Flore, and the inhibitory activity of blood coagulation factor shown by Apity time was Adams> Manchurian> Prosercer soup It appeared in the order of Ranger> Hopey> Flore, and showed the same pattern as the hot water extract. Therefore, it was confirmed that among the flower apple extracts, the hot water extract or ethanol extract of Adams, Prosper Sprenger, and Manchurian varieties can be developed as an antithrombotic agent, particularly, an anticoagulant capable of replacing aspirin with high side effects such as gastrointestinal disorders.

In addition, the anti-platelet activity of the flower apple hot water extract and the ethanol extract of the present invention was mainly found in the ethanol extract, in the order of Manchurian> Flore> Professor Sprenger> Adams variety. Therefore, it was confirmed that among the flower apple extracts, ethanol extracts of Adams, Flore, Prosercer Sprenger, and Manchurian varieties could be developed as antithrombotic agents, particularly antiplatelet agents, capable of replacing aspirin with high side effects such as gastrointestinal disorders.

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

The active ingredient of the present invention can be used for the 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, sensory abnormalities, personality changes, decreased vision, epilepsy seizures, pulmonary thrombosis , Deep vein thrombosis, swelling of the lower extremities, pain and acute peripheral artery occlusion, and intravenous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, brain venous sinus thrombosis and central retinal vein occlusion. In particular, the specific active ingredients described above of the present invention can also be used for the purpose of a blood coagulation inhibitor (anticoagulant) or a platelet aggregation inhibitor (antiplatelet agent).

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

The pharmaceutical composition may further include a carrier, excipient or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, and the like.

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

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

As a preferred embodiment, the formulation for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

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

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

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

“Subject” in the present invention includes, but is not particularly limited to, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits, or guinea pigs, for example. 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 beverages effective in preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, and dietary supplements, and may be used in the form of powders, granules, tablets, capsules, or beverages. .

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

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

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

As the flavoring agent, natural flavoring agents such as stevia such as taumatin, rebaudioside A or glycyrrhizine, and synthetic flavoring agents such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used in about 1 to 20 g, preferably about 5 to 12 g per 100 ml of health functional food of the present invention. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals, synthetic flavoring agents, flavoring agents such as natural flavoring agents, coloring agents and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloids It may contain a thickening agent, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the present invention will be described in more detail through examples. The following examples are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

[Example]

Example 1: Preparation of various flower apple extract and component analysis of the extract

In 2017, the flower apples cultivated and harvested at the Cheongsong-gun Agricultural Technology Center in Gyeongbuk were collected to remove foreign substances, and then sliced. Then, hot water and ethanol extracts were prepared. At this time, the flower apples used were the five most grown in the country: Adams, Hopey, Flore, Prosper Sprinkler, and Manchurian, and all of them used maturation fruits that produced more than 130 days after flowering (Table 1). And Figure 1).

[Table 1] Size and characteristics of various apples used in the experiment

On the other hand, the water content and physicochemical properties of the flower apple used and the color difference of the extract obtained by double hydrolysis are shown in Table 2.

[Table 2] Physicochemical Characteristics and Color Differences of Various Flower Apples Used in Experiment

As shown in Table 2, the average moisture content of the five apples was 74.5 ± 6.94%, which was 10-15% lower than that of the general apple. On the other hand, the average pH of flower apples was 3.46 ± 0.16, brix was 3.63 ± 0.62, and acidity was 0.445 ± 0.09. The highest brix was found in Flore (4.6%) and the highest acidity (0.49) in Adams and Manchurian. As a result of chrominance measurement, the highest brightness was found to be 21.47 in Manchurian, the redness was in Hoaei and the yellowness was highest in Adams. The overall color difference was 70.95 (Manchurian) to 76.55 (Hopeai), so it was easy to distinguish with the naked eye.

On the other hand, when preparing the ethanol extract, ethanol was added 20 times based on the weight of the flower apple, extracted once at room temperature, collected, filtered, and concentrated under reduced pressure to prepare a powder. Distilled water was added, extracted once at 100 ° C for 1 hour, and then the extract was collected and filtered, and then concentrated under reduced pressure to prepare a powder. Table 3 shows the results of each extraction efficiency and component analysis of the extract. Total polyphenols, total flavonoids, total sugars, and reducing sugars were determined by component analysis. The total polyphenol content was 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ saturated solution in 400 μl of the extracted sample solution, and allowed to stand at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. The total flavonoid content was extracted by stirring each sample with methanol for 18 hours, adding 4 ml of 90% diethylene glycol to 400 μl of the filtered extraction sample, adding 40 μl of 1 N NaOH again, and reacting at 37 ° C. for 1 hour to measure absorbance at 420 nm. Rutin was used as a standard reagent. The reducing sugar was quantified using the DNS method and the total sugar was phenol-sulfuric acid method.

[Table 3] Analysis of extraction efficiency and useful components of hot water extract and ethanol extract of various flower apples

As shown in Table 3, the extraction efficiency of hot water was 3.8 ~ 11.3%, which varied depending on the flower apple variety, but the extraction efficiency of ethanol was not higher than 3.4 ~ 4.5%. The average hot water extraction efficiency was 6.86 ± 2.95%, which was 1.7 times higher than the average ethanol extraction efficiency of 4.05 ± 0.36%, and overall, the total polyphenol content of the hot water extract was higher than that of the ethanol extract.

The highest polyphenol content among the five types of hot water extracts and ethanol extracts from the Flower Apple was found in Adams. In the case of hot water extracts, Adams> Hopey> Manchurian> Professor Sprinkler> Flore, and in the case of ethanol extracts, Adams >> Hopey> Flore> Manchurian> Prosper Sprinkler. This high polyphenol content is 5 to 10 times higher than that of commercial flower apple powder, and the flower apple extract was expected to exhibit various physiological activities. On the other hand, total sugar and reducing sugar contents were higher in Flore, Professor Sprinkler, and Manchurian.

Example 2: Blood clotting inhibitory activity of various flower apple extracts

The blood clotting inhibitory activity of the flower apple extracts of Example 1 was evaluated, and the results are shown in Tables 4 and 5. At this time, the blood clotting inhibitory activity evaluation method was evaluated according to the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14 509-513; Ryu et al. 2010. J. Life Science, 20. 922-928), thrombin time, prothrombin time and affinity time were measured. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China). Thrombin time, prothrombin time and apity time measurements were performed in the following procedure.

Thrombin Time

50 μl of 0.5U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ and 10 μl of sample extract of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) at 37 ° C. for 2 minutes, and then 100 μl of plasma was added. After that, the time until plasma clot was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of a sample as a solvent control. In the case of DMSO, the clotting time was 24.2 seconds. The thrombin inhibitory effect was expressed as the average value of the experiment repeated 3 or more times, and the thrombin inhibitory activity was expressed as the value of the solidification time when the sample was added divided by the solidification time of the solvent control.

Prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations are added to a tube of Amelung coagulometer KC-1A (Japan), heated at 37 ° C. for 3 minutes, then 130 μl of PT reagent is added and plasma is coagulated. Time until it 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 a sample as a solvent control. In the case of DMSO, a coagulation time of 16.8 seconds was shown. The prothrombin inhibitory activity was expressed as a value obtained by dividing the coagulation time when the sample was added by the coagulation time of the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of sample extract of various concentrations are added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37 ° C. for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) is added and again at 37 ° C. 3 Incubated for a minute. After 50μl CaCl ₂ (35mM) was added, the time until the plasma was clot was measured. DMSO was used as a solvent control instead of a sample, and in this case, a clotting time of 42.2 seconds was exhibited. The result of aPTT was expressed as the average value of the experiment repeated 3 times, and the blood coagulation factor inhibitory activity was expressed by dividing the aPTT at the time of adding the sample by the aPTT of the solvent control.

First, the results of measuring thrombin time, prothrombin time and apity time by adjusting each extract to a concentration of 5 mg / ml are shown in Table 4. The aspirin used as an active control prolonged thrombin time, prothrombin time, and apity time at a concentration of 5 mg / ml by 15 times or more compared to the additive-free group, and at a concentration of 1.5 mg / ml, extended by 1.42, 1.63, and 1.48 times, respectively. It showed anticoagulant activity. On the other hand, the flower apple hot water extract showed excellent anticoagulant activity in the Adams variety, and showed excellent activity in the Adams and Manchurian variety in the ethanol extract. In particular, the hot water extract of the Adams variety increased thrombin time, prothrombin time and apity time by 6.5,> 15 and> 15 times, respectively, at a concentration of 5 mg / ml, and ethanol extract thrombin at a concentration of 5 mg / ml. Time, prothrombin time and ap time were all increased by> 15 times compared to the additive-free group, and exhibited the same blood coagulation enzyme inhibitory and coagulation factor inhibitory activity as the commercial antithrombotic aspirin.

[Table 4] Blood coagulation inhibitory activity of flower apple extract (5mg / ml) by type

From the above results, the anticoagulant activity of each extract was evaluated by concentration, and the results are shown in Table 5. Finally, thrombin and prothrombin inhibitory activity of the flower apple hydrothermal extract was in the order of Adams> Prosercer Sprenger> Manchurian> Hopei> Flore, and the inhibitory activity of blood coagulation factor in Apity time was Adams> Manchurian> Professor It was almost the same as Sprenger> Hopey> Flore. In addition, thrombin and prothrombin inhibitory activity among the anticoagulant activity of the flower apple ethanol extract was in the order of Adams> Prosper Sprenger> Manchurian> Hopei> Flore, and the inhibitory activity of blood coagulation factor shown by Apity time was Adams> Manchurian> Prosercer soup It appeared in the order of Ranger> Hopey> Flore, and showed the same pattern as the hot water extract. Therefore, it was confirmed that Adams, Prosper Sprenger, and Manchurian varieties among the flower apple extracts can be developed as antithrombotic agents that can replace aspirin, which is highly concerned about side effects such as gastrointestinal disorders, particularly blood coagulation inhibitors (anticoagulants).

[Table 5] Blood coagulation inhibitory activity of flower apple extract by concentration

Example 3: Platelet aggregation inhibitory activity of various flower apple extracts

Table 1 shows the results of evaluating the activity of inhibiting human platelet aggregation of various flower apple extracts of Example 1. Platelets are discoid small cells that circulate blood vessels with various blood cells, but instead of nucleus, they have cytoplasmic granules containing high concentrations of various substances related to vascular damage protection and platelet aggregation, and aggregation when damage to the vascular lining appears It is an important cell that secretes factors and combines with collagen exposed due to damage to endothelial cells to form a primary hemostatic plug to initiate thrombus production. Therefore, inhibition of platelet aggregation is a very important activity that prevents thrombus production. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Human platelets were used as platelets, which were washed once with washing buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 1 mM EDTA, pH 6.5). Thereafter, after resuspending in suspending buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 0.49mM MgCl ₂ , 0.25% gelatin, pH 7.4), 10 min at 3,000rpm After centrifugation, the suspension was re-suspended in a suspending buffer, and the platelet number was adjusted to be 4x10 ⁹ / ml. Thereafter, 2.5 μl collagen was added to the 1 ml suspension and reacted for 5 minutes, and platelet aggregation was measured at 37 ° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 6] Platelet aggregation inhibitory activity of various flower apple extracts

As shown in Table 6, first, aspirin showed a strong platelet aggregation inhibition (aggregation degree 21.5-62.7%) in a concentration-dependent manner at a concentration of 0.125 to 0.25 mg / ml, indicating the basis for clinical use as an antithrombotic agent. On the other hand, hot water extracts of the flower apple did not affect human platelet aggregation at a concentration of 0.25 mg / ml, and exhibited weak aggregation promoting activity. The flower apple ethanol extracts showed good inhibition of platelet aggregation in 4 species except the Hopei variety at a concentration of 0.25 mg / ml, and the Floret and Manchurian varieties showed agglomeration of 63.5 to 65.6% compared to no additives, aspirin 0.125 mg The aggregation inhibition was similar to the / ml concentration. The Adams variety showed 92.7% cohesiveness and showed weak platelet aggregation inhibitory activity. Accordingly, among the apple ethanol extracts, Adams, Flore, Prosercer Prorenger, and Manchurian varieties suggest that it can be developed as an antithrombotic agent, in particular, a platelet aggregation inhibitor (antiplatelet agent) as a platelet aggregation inhibitory activity.

Example 4: Human red blood cell hemolytic activity of various flower apple extracts

Flower apples have been processed and processed into 쨈, vinegar and liquor for a long time, and are currently registered as food raw materials in Korea to ensure safety. In the present invention, human red blood cell hemolytic activity was evaluated to evaluate the acute toxicity of the flower apple extract, and the results are shown in Table 7. At this time, the hemolytic activity was evaluated according to an existing report (Kim Mi-sun et al., 2014. J. Life Sci. 24: 515-521). Simply, 100 μl of human red blood cells washed three times with PBS were added to a 96-well microplate and various After adding 100 μl of the sample solution of the concentration and reacting for 30 minutes at 37 ° C., the reaction solution was centrifuged (1,500 rpm) for 10 minutes to transfer 100 μl of the supernatant to a new microtiter plate, and the degree of hemoglobin outflow due to hemolysis was measured at 414 nm. Did. 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 7] Human erythrocyte hemolysis activity of various flower apple extracts

As a result, as shown in Table 7, DMSO and water used as controls did not have hemolytic activity, and triton X-100 confirmed that hemolysis of 100% of red blood cells was performed at a concentration of 1 mg / ml. In addition, it was confirmed that the antifungal agent empoteracin B, which is reported to exhibit hemolytic activity, hemolytically erythrocytes 93.7% even at a concentration of 0.05 mg / ml. On the other hand, the flower apple extract had no hemolytic activity in both the hot water extract and the ethanol extract. Therefore, it is judged that the flower apple hot water and ethanol extract will not exhibit the problem of causing acute toxicity separately.

Example 5: Plasma, acid and thermal stability evaluation of flower apple extract

Plasma stability, thermal stability and acid stability for antioxidant activity were confirmed for the flower apple extract obtained in Example 1 above. The samples exhibited high stability due to no decrease in blood coagulation inhibition and platelet aggregation inhibitory activity even after 1 hour heat treatment at 100 ° C, 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma. Therefore, the extracts are expected to maintain excellent antithrombotic activity during digestion and absorption and food production.

Claims (4)

A pharmaceutical composition for the prevention or treatment of thrombosis, which contains the flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient. The pharmaceutical composition according to claim 1, wherein the flower apple extract is a hot water or ethanol extract of a flower apple variety selected from the group consisting of Adams, Prosercer Sprinkler, and Manchurian. The pharmaceutical composition of claim 1, wherein the flower apple extract is an ethanolic extract of Flore variety. A health functional food for preventing or improving thrombosis comprising the active ingredient according to any one of claims 1 to 3.

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