KR102155912B1 - 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 the prevention or treatment of thrombosis containing an extract of Malus prunifolia Wild. Borkh as an active ingredient, and more specifically, an extract of the mature fruit of the flower apple It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through strong blood coagulation inhibition and platelet coagulation inhibition using as an active ingredient, and a health functional food. The flower apple extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and a health functional food exhibits strong antithrombotic activity through inhibition of blood clotting factors and blood clotting factors related to thrombosis and inhibiting platelet aggregation. Since it does not show any hemolytic activity against red blood cells, has excellent thermal stability, and does not lose blood coagulation and platelet coagulation inhibitory effects even in acidic conditions of pH 2 and plasma, thrombosis such as ischemic stroke and hemorrhagic stroke through improved blood circulation It is expected that it can be used as a pharmaceutical composition and health functional food for the prevention and treatment/improvement of medicine, and the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc., and can be prepared in a form that can be taken at any time. Since it is effective, it is a very useful invention in the pharmaceutical and food industries.

Description

Pharmaceutical composition and health functional food for the prevention or treatment of thrombosis containing flower apple extract as an active ingredient {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 and a health functional food for the prevention or treatment of thrombosis containing an extract of Malus prunifolia Wild. Borkh as an active ingredient, and more specifically, an extract of the mature fruit of the flower apple It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through strong blood coagulation inhibition and platelet coagulation inhibition using as an active ingredient, and a health functional food.

Blood as a constituent of the human body has various important functions such as transporting and buffering oxygen, nutrients, and waste products, maintaining body temperature, controlling osmotic pressure and maintaining ionic balance, maintaining moisture schedule, controlling liquid properties, maintaining and controlling blood pressure, and protecting 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.

On the other hand, the formation of fibrin thrombi causes thrombin, which is involved in fibrin coagulation, to be activated through several step reactions of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen, and fibrin monomers are polymerized by calcium, and platelets and It binds to endothelial cells and forms a fibrin polymer 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 substance inhibiting the activity of thrombin 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 expensive, but also bleeding side effects, gastrointestinal disorders and irritability. Its use is limited due to reactions and the like.

On the other hand, the flower apple is a deciduous broad-leaved tree belonging to the Rosaceae apple tree genus (Malus), and its origin is Europe, Asia, and North America, and is called Flower Apple, Plum-leaved Apple, Chines Apple, Crab Apple in English. Flower apples grow very well in barren soil, and are used as the rootstock of apple trees that are difficult to self-fertilize because they bloom a large amount of flowers at once, and because of their gorgeous flowers, they are also used as an ornamental tree.

The fruit of the flower apple is much smaller than that of ordinary apples, and is rich in fructose, glucose, tartaric acid, and vitamin C, so it is known to be good for maintaining health, relieving fatigue, and constipation. They are not used, and most of them are naturally discarded due to lack of verification power. In the private sector, when cooking meat, it was also used as a softener to soften the meat, and drinking with flower apples was used as a medicine because it has the effect of relieving fatigue, increasing appetite, nervousness, insomnia, and constipation. In oriental medicine, dried fruit is used for dry stomach, indigestion, loss of appetite, acid deficiency or hyperacidity, and is known to be used as medicine for diarrhea, menstrual pain, frostbite, stomach, low back pain, and intestinal bleeding.

Flower apples are characterized by free cross-species or intra-species hybridization due to no genetic barrier, and over 700 varieties have been developed worldwide. The color of the flower is white, light pink, pink, deep pink, crimson, etc. The flowering period is 8-12 days in April, and most of the fruits are known to have a diameter of 2.5cm or less (Dongyoung So. 2011, Konkuk University's master's thesis). ). In addition, flower apples are characterized by strong cold resistance, excellent environmental adaptability, and growing well in barren soil. In the case of apple trees, flowering time is different for each cultivar, and there are differences in pests and cultivation methods, so pollination for each cultivated cultivar is required.Manchurian, Hopaei, Adams, and Professor Sprenzer are the most cultivated flower apple varieties in Korea. , Floret, SKK-14, and SKK-16.

In general, in the private sector, the fruit of the hawthorn (Chinese hawthorn Crataegus cuneata Sieb, Western hawthorn Crataegus oxyacantha L.) is often called confused because it is similar to the flower apple.

As for research related to flower apples, selection of flower apples suitable for pollination by apple varieties (In-Gyu Kang et al., 2002. Journal of Horticultural Science and Technology 20: 330-334; Kwang-Min Son et al., 2013. Protected Horticulture and Plant Factory, 22: 116- 122), a study on the reason why it grows well in barren soil (Huang L. et al., 2018, Plant Physiol Biochem. 127:185-193; Tan Y et al., 2017, Plant Physiol Biochem. 115: 219-228) is mainly conducted. It has been reported that the anthocyanin pigment of the flower apple has excellent light resistance and heat resistance, making it easy to use as a natural red pigment resource (Kim Yong-hwan, 1999, Korean J. Food Nutr. 12: 85-90). However, until now, studies on useful components and useful physiological activities of flower apples are very limited.

Recently, it has been reported that the polyphenols and flavonoids of flower apples are more than twice that of ordinary apples, and are rich in tartaric acid and vitamin C (Dongyoung So. 2011, Master's thesis at Konkuk University), and gallic acid, protocatechuic than ordinary apples. It has been reported that the content of phenolic acids such as acid, chlorogenic acid, p-coumaric acid, and ferulic acid is high, and the content of flavonoids such as quercetin and myricetin is high, so that it exhibits better antioxidant activity than apples (KM Maria John et al., 2014. Food. Chemistry 163: 46-50). In addition, recently, the flower apple Malus'Red jade', Malus hupehensis (Pamp.) Rehd. And Malus prunifolia (Willd.) Borkh 3 species citric acid, p-coumaric acid, hyperoside, myricetin, naringenin, quercetin, kampferol, gentiopicroside, ursolic acid and 8-epiloganic acid were isolated, 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 potent anticoagulant and platelet-inhibitory activity of the flower apple extract has not been known.

Patents related to flower apples are disclosed in Korean Patent Registration No. 10-1081059 [a flower mixture extract having antioxidant and whitening activity and its extraction method, and a cosmetic composition containing the flower mixture extract] and Japanese Patent JP-0137455 [ Production of nursery stock of pear tree] has been disclosed, and patents or research results for active extracts of non-toxic flower apples are known, while showing strong antithrombotic activity through potent blood coagulation inhibition and platelet aggregation inhibition of flower apples. There is no bar.

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

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 preventing or treating thrombosis containing a flower apple extract as an active ingredient and a health functional food I want to.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing a 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 cultivar selected from the group consisting of Adams, Professor Sprenger and Manchurian.

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

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing a 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 cultivar selected from the group consisting of Adams, Professor Sprenger and Manchurian.

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

The flower apple extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and a health functional food exhibits strong antithrombotic activity through inhibition of blood clotting factors and blood clotting factors related to thrombosis, and at the same time, human Since it does not show any hemolytic activity against red blood cells, has excellent thermal stability, and does not lose blood coagulation and platelet coagulation inhibitory effects even in acidic conditions of pH 2 and plasma, thrombosis such as ischemic stroke and hemorrhagic stroke through improved blood circulation It is expected to be used as a pharmaceutical composition and health functional food for the prevention and treatment/improvement of the drug. The active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc., so that it can be prepared into a form that can be taken at any time. Since it is effective, it is a very useful invention in the pharmaceutical and food industries.

1 shows a flower apple variety used in an example of the present invention, and shows Adams, Hopaey, Floret, Professor Sprenger, and Manchurian varieties from the left.

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of a flower apple, the inventors of the present invention prepare a hot water and ethanol extract by harvesting flower apples to remove foreign substances, and evaluate the activity to test the antithrombotic activity. The apple extract was confirmed, and the active extract did not show any hemolytic activity against human red blood cells, but it was confirmed that it had excellent heat stability and acid stability, so that the flower apple extract was used for the prevention or treatment/improvement of thrombosis. It was intended to be used as a composition and health functional food.

Specifically, in order to develop a pharmaceutical composition and a health functional food for preventing or treating/improving thrombosis using flower apples used for various purposes in the private sector, the present inventors have developed a variety of mature flower apples (Adams, Hopa A , Floret, Professer Sprenger, Manchurian), respectively, prepared hot water extract and ethanol extract, and their antithrombotic activity was directly inhibited in human plasma and human thrombin (Thrombin Time), prothrombin inhibition (Prothrombin) Time) and activated Partial Thromboplastin Time (aPTT) were evaluated. As a result, strong anticoagulant activity was confirmed in hot water extracts and ethanol extracts of Adams, Professor Sprenger and Manchurian cultivar among flower apples. This antithrombotic activity was a potent antithrombotic activity that surpassed the 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 ethanol extracts of Adams, Floret, Professer Sprenger and Manchurian cultivar were excellent in inhibiting platelet aggregation. In addition, it was confirmed that neither of the flower apple extracts showed hemolytic activity against human red blood cells.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing a 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 cultivar selected from the group consisting of Adams, Professor Sprenger and Manchurian.

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

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing a 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 cultivar selected from the group consisting of Adams, Professor Sprenger and Manchurian.

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

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

The present invention comprises the steps of harvesting and removing foreign substances from mature apples of various varieties; Slicing the flower apple and preparing an extract; Evaluating antithrombotic activity of flower apple extract; And investigating the stability of the active extract.

The "flower apple" of the present invention refers to a mature flower apple in which an ovary is generated after 130 days or more after flowering. In the present invention, the flower apples of Adams, Hopaey, Floret, Professor Sprenger and Manchurian varieties were used.

The "flower apple extract" included in the composition of the present invention comprises the steps of pulverizing the mature flower apple; Extracting the flower apple crushed product with an organic solvent; Filtering the extract using a filter net of 0.06 mm or less; And it can be obtained by the step of concentrating 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, ethyl acetate, etc.), and a mixed solvent of the lower alcohol and water Etc. may be used, and hot water or 95% ethanol extraction is most preferred.

Among the anticoagulant activities of the hot water extract of the flower apple of the present invention, the inhibitory activities of thrombin and prothrombin were in the order of Adams> Professor Sprenger> Manchurian> Hopa A> Flore, and the blood coagulation factor inhibitory activity indicated by AP Time was Adams> Manchurian> They were similar in the order of Professor Sprenger> Hopa A> Floret. In addition, thrombin and prothrombin inhibitory activity among the anticoagulant activities of the ethanol extract of flower apple were in the order of Adams> Profesor Sprenger> Manchurian> Hopa A> Flore. It appeared in the order of Ranger>Hopaei>Flore, and showed the same pattern as the hot water extract. Therefore, it was confirmed that the hot water extract or ethanol extract of Adams, Profesor Sprenger, and Manchurian cultivar among the flower apple extracts can be developed as antithrombotic agents, especially anticoagulants, capable of replacing aspirin, which has a high risk of side effects such as gastrointestinal disorders.

In addition, the antiplatelet activity of the hot water extract and ethanol extract of the flower apple of the present invention was mainly found in the ethanol extract, in the order of Manchurian> Floret> Professor Sprenger> Adams variety. Therefore, it was confirmed that the ethanol extracts of Adams, Floret, Professer Sprenger and Manchurian cultivar among the flower apple extracts can be developed as antithrombotic agents, especially antiplatelet agents that can replace aspirin, which is highly likely to have side effects such as gastrointestinal disorders.

The flower apple extract of the present invention may be prepared into a powder through a conventional powdering process such as vacuum drying, freeze drying or spray drying. They are not degraded by various degrading enzymes in plasma, and they maintain their 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 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. In particular, the above-described specific active ingredient of the present invention may be used as a blood coagulation inhibitor (anticoagulant) or platelet aggregation inhibitor (antiplatelet agent).

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, which 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.

As the flavoring agent, natural flavoring agents such as taumatin, rebaudioside A, or stevia such as glysirhizin, 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 various flower apple extracts and analysis of components of the extract

Flower apples cultivated and harvested at the Agricultural Technology Center in Cheongsong-gun, Gyeongbuk in 2017 were recovered, removed foreign substances, and then minced, and then hot water and ethanol extracts were prepared. The flower apples used at this time were the five species of Adams, Hopaei, Floret, Professor Spranger and Manchurian, which are the most cultivated in Korea, and all of them used mature fruits with ovaries over 130 days after flowering (Table 1). And FIG. 1).

[Table 1] Size and characteristics of various flower 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 watering are shown in Table 2.

[Table 2] Physicochemical properties and color differences of various flower apples used in the experiment

As shown in Table 2, the average moisture content for the five flowering apples was 74.5±6.94%, which was 10-15% lower than that of ordinary apples. Meanwhile, 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) was found in Adams and Manchurian. As a result of measuring the color difference, the highest brightness was 21.47 in Manchurian, and the redness was highest in Hopa-A, and the yellowness was highest in Adams. The overall color difference ranged from 70.95 (Manchurian) to 76.55 (Hopa A), making it easy to distinguish with the naked eye.

On the other hand, when preparing the ethanol extract, 20 times the weight of the flower apple was added, extracted once at room temperature, the extract was collected and filtered, and then concentrated under reduced pressure to prepare a powder, and the hot water extract was 20 times the weight of the flower apple. Distilled water was added, and extracted once at 100° C. for 1 hour, the extract was collected, filtered, and concentrated under reduced pressure to prepare a powder. Each extraction efficiency and component analysis results of the extract are shown in Table 3. The content of total polyphenols, total flavonoids, total sugars and reducing sugars was measured by component analysis. 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, 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 1 hour reaction at 37°C. 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 hot water extract and ethanol extract of various flower apples

As shown in Table 3, the hot water extraction efficiency was 3.8~11.3%, which was varied depending on the flower apple cultivar, but the ethanol extraction efficiency was 3.4~4.5%, showing no significant difference. The average hot water extraction efficiency was 6.86±2.95%, about 1.7 times higher than the average ethanol extraction efficiency 4.05±0.36%, and the total polyphenol content of the hot water extract was higher than that of the ethanol extract.

The highest polyphenol content was found in Adams among the five hot-water extracts and ethanol extracts of the flower apple, and in the hot-water extract, Adams> Hopaei> Manchurian> Professor Sprenger> Floret was found in the order of Adams. >> Hopaey> Floret> Manchurian> Professor Spranger. This high polyphenol content is 5 to 10 times higher than that of commercial flower apple powder, and flower apple extract is expected to exhibit various physiological activities. On the other hand, the content of total sugar and reducing sugar was high in Floret, Profesor Spreader, and Manchurian.

Example 2: Anticoagulant activity of various flower apple extracts

The blood coagulation 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 method for evaluating the blood coagulation inhibitory activity 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 AP time were measured by 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 extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) at 37°C for 2 minutes to react, and then 100 μl 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 24.2 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

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solution of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37°C for 3 minutes, and then 130 μl of PT reagent was added and the 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 16.8 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 then 3 at 37°C. Incubated for minutes. After that, 50 μl CaCl ₂ (35 mM) was added and the time until plasma coagulated was measured. DMSO was used instead of the sample as a solvent control, and in this case, the coagulation time was 42.2 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.

First, each extract was adjusted to a concentration of 5 mg/ml, and the results of measuring thrombin time, prothrombin time, and AP time are shown in Table 4. Aspirin used as an active control extended thrombin time, prothrombin time, and AP time by more than 15 times compared to the non-added group at a concentration of 5 mg/ml, and at 1.5 mg/ml concentration, it was excellent by extending 1.42, 1.63 and 1.48 times, respectively. It showed anticoagulant activity. On the other hand, the hot water extract of the flower apple showed excellent anticoagulant activity in the Adams cultivar, and the ethanol extract showed excellent activity in the Adams and Manchurian cultivars. In particular, the hot water extract of the Adams variety increased thrombin time, prothrombin time, and AP time by 6.5, >15 and >15 times, respectively, at a concentration of 5mg/ml compared to the non-added food, and the ethanol extract increased thrombin at a concentration of 5mg/ml. Time, prothrombin time, and AP time were all increased >15 times compared to the non-added food, showing the same blood coagulation enzyme inhibition and coagulation factor inhibitory activity as commercial antithrombotic aspirin.

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

From the above results, the anticoagulant activity by concentration of each extract was evaluated, and the results are shown in Table 5. Finally, among the anticoagulant activities of hot water extract of flower apple, the inhibitory activities of thrombin and prothrombin were in the order of Adams> Profesor Sprenger> Manchurian> Hopa A> Flore. They were almost similar in the order of Sprenger> Hopaey> Floret. In addition, thrombin and prothrombin inhibitory activity among the anticoagulant activities of the ethanol extract of flower apple were in the order of Adams> Profesor Sprenger> Manchurian> Hopa A> Flore. It appeared in the order of Ranger>Hopaei>Flore, and showed the same pattern as the hot water extract. Therefore, it was confirmed that Adams, Profesor Sprenger and Manchurian varieties among the flower apple extracts can be developed as antithrombotic agents, especially blood coagulation inhibitors (anticoagulants), which can replace aspirin, which has a high risk of side effects such as gastrointestinal disorders.

[Table 5] Anticoagulant Activity of Flower Apple Extracts by Concentration

Example 3: Platelet Aggregation Inhibitory Activity of Various Flower Apple Extracts

The human platelet aggregation inhibitory activity of the various flower apple extracts of Example 1 was evaluated and the results are shown in Table 6. Platelets are disk-shaped small cells that circulate in blood vessels along 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 a high concentration, and aggregation when damage to the inner wall of blood vessels occurs. It is an important cell that secretes factors and forms a primary hemostatic plug by binding to collagen, etc. exposed due to damage to endothelial cells to initiate thrombus formation. Therefore, inhibition of platelet aggregation 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 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 strong platelet aggregation inhibition (aggregation degree 21.5-62.7%) in a concentration-dependent manner at a concentration of 0.125-0.25mg/ml, indicating the basis for its use as an antithrombotic agent in clinical practice. On the other hand, the hot water extracts of the flower apple did not affect human platelet aggregation at the concentration of 0.25mg/ml, and showed weak aggregation promoting activity. Ethanol extracts of flowering apples showed good platelet aggregation inhibition in 4 species excluding Hopaei cultivar at a concentration of 0.25mg/ml, and Floret and Manchurian cultivar showed a degree of aggregation of 63.5~65.6% compared to the non-added cultivar, 0.125mg aspirin. Aggregation inhibition similar to /ml concentration was shown. Adams variety showed a degree of aggregation of 92.7%, showing weak platelet aggregation inhibitory activity. Therefore, it has been suggested that Adams, Floret, Professer Prorenzer and Manchurian cultivars among the ethanol extracts of flower apples can be developed as antithrombotic agents, especially platelet aggregation inhibitors (antiplatelet agents) with their platelet aggregation inhibitory activity.

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

Flower apples have long been processed and edible into jam, vinegar and alcoholic beverages, and are currently registered as a food ingredient in Korea, ensuring safety. In the present invention, in order to evaluate the acute toxicity of the flower apple extract, human red blood cell hemolytic activity was evaluated, and the results are shown in Table 7. At this time, the hemolytic activity was evaluated according to the previous report (Miseon Kim et al., 2014. J. Life Sci. 24: 515-521), and simply 100 μl of human red blood cells washed three times with PBS was added to a 96-well microplate and various After adding 100μl of the sample solution of concentration, the reaction was carried out at 37°C for 30 minutes, and then, the reaction solution was centrifuged for 10 minutes (1,500rpm), and 100μl of the supernatant was transferred to a new microtiter plate, and the degree of hemoglobin leakage due to hemolysis was measured at 414nm. I did. 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 7] Human red blood cell hemolytic activity of various flower apple extracts

As a result, as shown in Table 7, it was confirmed that DMSO and water used as controls did not have hemolytic activity, and triton X-100 hemolytic 100% red blood cells at a concentration of 1 mg/ml. In addition, it was confirmed that the antifungal agent Empoteracin B, reported to exhibit hemolytic activity, hemolytic 93.7% of red blood cells even at a concentration of 0.05 mg/ml. Meanwhile, the flower apple extract had no hemolytic activity in both the hot water extract and the ethanol extract. Therefore, it is judged that the hot water and ethanol extracts of flower apples will not show a separate problem of inducing acute toxicity.

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

Plasma stability, thermal stability, and acid stability against antioxidant activity were confirmed for the flower apple extract obtained in Example 1 above. The samples 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, but did not inhibit blood coagulation and inhibit platelet aggregation, indicating high stability. Therefore, the above extracts are expected to maintain excellent antithrombotic activity during digestion and absorption and during food manufacturing.

Claims (4)

A pharmaceutical composition for the prevention or treatment of thrombosis containing ethanol extract of Floret cultivar or Manchurian cultivar ( Malus prunifolia Wild. Borkh) as an active ingredient. delete delete A health functional food for preventing or improving thrombosis containing ethanol extract of Floret variety or Manchurian variety flower apple ( Malus prunifolia Wild. Borkh).

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