KR102501812B1 - Pharmaceutical composition comprising the extraction of flower apple skk14 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 SKK14 variety Malus prunifolia Wild. Borkh as an active ingredient, and more specifically, to It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis through strong blood coagulation inhibition and platelet aggregation inhibition using an extract of immature or mature fruit of flowering apple as an active ingredient. The pharmaceutical composition for preventing or treating thrombosis of the present invention and the SKK14 variety flower apple extract as an active ingredient of the health functional food exhibit strong antithrombotic activity through inhibition of thrombosis-related enzymes and blood coagulation factors and inhibition of platelet aggregation, At the same time, it does not show any hemolytic activity on human red blood cells, has excellent thermal stability, and does not show loss of blood coagulation inhibitory and platelet aggregation inhibitory effects even in acidic conditions of pH 2 and plasma, so ischemic stroke and hemorrhagic stroke by improving blood circulation It is expected that it can be used as a pharmaceutical composition for preventing and treating/improving thrombosis and as a health functional food, and the active ingredient is processed into various forms such as extracts, powders, pills, and tablets to be prepared in a form that can be taken at all times. It is a very useful invention in the pharmaceutical and food industries because it has excellent effects that can be obtained.

Description

PHARMACEUTICAL COMPOSITION COMPOSITION THE EXTRACTION OF FLOWER APPLE SKK14 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 an extract of SKK14 variety Malus prunifolia Wild. Borkh as an active ingredient, and more specifically, to It relates to a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis through strong blood coagulation inhibition and platelet aggregation inhibition using an extract of immature or mature fruit of flowering apple as an active ingredient.

Blood, as a component of the human body, has various important functions such as oxygen, nutrients, and waste transport and buffering, body temperature maintenance, osmotic pressure control and ion balance maintenance, water constant maintenance, fluid control, blood pressure maintenance and control, and biological defense. there is. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the clot dissolution reaction system are mutually controlled in the body. , It has been reported that the blood coagulation system is activated to form a fibrin clot centered on platelet aggregates.

On the other hand, the generation of fibrin clots goes through a multi-step reaction of numerous blood coagulation factors, and thrombin involved in fibrin coagulation is activated to finally produce fibrin monomers from fibrinogen, which are polymerized by calcium to form platelets and platelets. It binds to endothelial cells and forms a permanent blood clot while forming a fibrin polymer cross-linked by factor XIII. In addition, thrombin plays a pivotal role in the formation of blood clots, such as promoting blood coagulation by activating platelets, factor V, and factor VII. Therefore, a substance that inhibits the activity of thrombin can be used as a very useful preventive and therapeutic agent for various thrombotic diseases caused by abnormal blood coagulation. On the other hand, in the endogenous thrombogenesis pathway, it is known that sequential activation of factor XII, factor XI, factor IX, and factor X followed by activation of prothrombin finally activates thrombin, and specific inhibition of blood coagulation factors is also important for the development of therapeutic agents for thrombotic diseases. becoming a target Until now, various anticoagulants such as heparin, coumarin, aspirin, and urokinase, anticoagulants, antiplatelet agents, and thrombolytic agents have been used for the prevention and treatment of thrombotic diseases. Its use is limited due to reactions and the like.

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

The fruits of flower apples are much smaller than ordinary apples and are rich in fructose, glucose, tartaric acid, and vitamin C, and are known to be good for health maintenance, fatigue recovery, and constipation. It is not used, and most of them are naturally dropped and discarded due to lack of harvesting ability. In the private sector, it was also used as a meat tenderizer to soften meat when cooking meat, and drinking it as a medicinal drink was effective in relieving fatigue, increasing appetite, nervousness, insomnia, and constipation. In oriental medicine, dried fruit is used for dry stomach, indigestion, anorexia, gastric acid deficiency or hyperacidity, and is known to be used as a medicine for diarrhea, menstrual pain, frostbite, stomachache, back pain, and intestinal bleeding.

Flower apples have no genetic barriers and are characterized by free hybridization between species or within species, and about 700 varieties have been developed worldwide. There are various colors of flowers such as white, light pink, pink, dark pink, and crimson, and the flowering period is about 8 to 12 days, and it blooms in April. ). In addition, flower apples have strong cold resistance, excellent environmental adaptability, and are characterized by growing well in barren land. In the case of apple trees, pollination is required for each cultivar due to differences in flowering time, pests and diseases and cultivation methods for each cultivar. , Flore, SKK-14, SKK-16, etc.

Usually, in the private sector, the fruits of hawthorn (Chinese hawthorn Crataegus cuneata Sieb, Western hawthorn Crataegus oxyacantha L.) are similar to flower apples, so they are often confusedly called, but this should be classified as incorrect.

For research related to flower apples, selection of flower apples suitable for pollination by apple variety (Kang In-gyu et al., 2002. Journal of Horticultural Science and Technology 20: 330-334; Son Gwang-min et al., 2013. Protected Horticulture and Plant Factory, 22: 116- 122), and studies on why it grows well even 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) are mainly in progress. It has been reported that the anthocyanin pigment of flower apple has excellent light resistance and heat resistance, so it can be easily used as a natural red pigment resource (Kim Yong-hwan, 1999, Korean J. Food Nutr. 12: 85-90). However, to date, studies on useful components and useful physiological activities of flower apples have been very limited.

Recently, it has been reported that the polyphenol and flavonoid components of flower apples are about twice as high as those of ordinary apples, and that they are rich in tartaric acid and vitamin C (So Dong-young. 2011, Konkuk University master's thesis), and gallic acid and protocatechuic acid content are higher than those of 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 also high, indicating superior antioxidant activity than apples (KM Maria John et al., 2014. Food Chemistry 163: 46-50). Also, recently, flower apple Malus 'Red jade', Malus hupehensis (Pamp.) Rehd. and citric acid, p-coumaric acid, hyperoside, myricetin, naringenin, quercetin, kampferol, gentiopicroside, ursolic acid and 8-epiloganic acid from 3 species of Malus prunifolia (Willd.) Borkh to lower cholesterol and prevent hyperlipidemia has been reported (Chao Wen et al., 2018. J. Pharmaceutical Biomedical Analysis 150: 144-151). However, until now, the strong blood coagulation inhibitory and platelet aggregation inhibitory activity of flower apple extract has not been known.

Patents related to flower apples include Korean Registered Patent No. 10-1081059 [Flower mixture extract having antioxidant and whitening activity, extraction method thereof, and cosmetic composition containing the flower mixture extract] and Japanese Patent JP-0137455 [ Production of nursery stock of pear tree] has been disclosed, and there are no known patents or research results for active extracts of flower apples that exhibit strong antithrombotic activity through strong inhibition of blood coagulation and inhibition of platelet aggregation, and are non-toxic. no bar

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

The present invention was made to solve the problems of the prior art as described above, and the problem to be solved in the present invention is a pharmaceutical composition for preventing or treating thrombosis containing an extract of SKK14 variety of apple blossom as an active ingredient and a health function to provide food.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing SKK14 variety of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple of the SKK14 variety is preferably an immature fruit.

The extract is preferably a hot water extract.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing SKK14 variety of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple of the SKK14 variety is preferably an immature fruit.

The extract is preferably a hot water extract.

The pharmaceutical composition for preventing or treating thrombosis of the present invention and the SKK14 variety flower apple extract as an active ingredient of the health functional food exhibit strong antithrombotic activity through inhibition of thrombosis-related enzymes and blood coagulation factors and inhibition of platelet aggregation, At the same time, it does not show any hemolytic activity on human red blood cells, has excellent thermal stability, and does not show loss of blood coagulation inhibitory and platelet aggregation inhibitory effects even in acidic conditions of pH 2 and plasma, so ischemic stroke and hemorrhagic stroke by improving blood circulation It is expected that it can be used as a pharmaceutical composition for preventing and treating/improving thrombosis and as a health functional food, and the active ingredient is processed into various forms such as extracts, powders, pills, and tablets to be prepared in a form that can be taken at all times. It is a very useful invention in the pharmaceutical and food industries because it has excellent effects that can be obtained.

Figure 1 shows flower apples of immature SKK14 cultivar 70 days after flowering.
Figure 2 shows the flower apples of the mature SKK14 cultivar 130 days after flowering.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, in order to test the antithrombotic efficacy of flower apples, harvested various flower apples, removed foreign substances, prepared an extract from them in a certain way, and evaluated the activity to obtain SKK14 varieties exhibiting antithrombotic activity. The flower apple extract was confirmed, and the active extract did not exhibit any hemolytic activity on human red blood cells, but it was confirmed that it had excellent thermal stability and acid stability, thereby making the flower apple extract a pharmaceutical for preventing or treating/improving thrombosis It was intended to be used as an enemy composition and health functional food.

Specifically, in order to develop a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis using flower apples, which are used for various purposes in the private sector, the present inventors harvested flower apples of various varieties and then hot watered them, respectively. Extracts were prepared, and their antithrombotic activity was evaluated for direct thrombin inhibition (Thrombin Time), prothrombin inhibition (Prothrombin Time) and activated partial thromboplastin time (aPTT) for human plasma and human thrombin. did As a result, the strong anticoagulant activity was confirmed in the flower apple extract of the SKK14 variety. This antithrombotic activity was a strong antithrombotic activity that surpassed that of aspirin, which is used as an antithrombotic agent in clinical practice. In addition, as a result of evaluating the human platelet aggregation inhibitory ability of the extract, very excellent platelet aggregation inhibitory ability was confirmed in immature fruits. In addition, it was confirmed that the extract did not show hemolytic activity against human red blood cells.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing SKK14 variety of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple of the SKK14 variety is preferably an immature fruit.

The extract is preferably a hot water extract.

In addition, the present invention provides a health functional food for preventing or improving thrombosis containing SKK14 variety of flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient.

The flower apple of the SKK14 variety is preferably an immature fruit.

The extract is preferably a hot water extract.

Hereinafter, the manufacturing method and efficacy test of the flower apple extract of the SKK14 variety of the present invention will be described in more detail.

The present invention includes the steps of harvesting flower apples of various varieties and removing foreign substances; Slicing flower apples and preparing an extract; Antithrombotic activity evaluation step of flower apple extract; and a step of examining the stability of the active extract.

The "flower apple of the SKK14 variety" of the present invention may be an immature fruit that does not produce an ovary 70 days after flowering or a mature fruit that produces an ovary 130 days or more after flowering. The "SKK14" variety is a variety that has been applied for variety name registration at the National Seed Resources of Korea and is registered as variety name registration number 03-0001-151 on October 15, 2014.

The "flower apple extract" included in the composition of the present invention comprises the steps of grinding immature or mature flower apples; Extracting the pulverized flower apples with an organic solvent; Filtering the extract using a filter net of 0.06 mm or less; And it can be obtained by concentrating it 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, 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.

In the present invention, as a result of measuring the thrombin time, prothrombin time and AP time with the hot water extract of immature flower apples of the SKK14 variety at a concentration of 5 mg/ml, it was confirmed that all of them were extended more than 15 times compared to the non-additive group, and 2.5 mg/ml Even at the ml concentration, it was extended by 1.87, 3.69, and 1.17 times, respectively, indicating excellent thrombin, prothrombin, and coagulation factor inhibition compared to aspirin. In addition, as a result of measuring the thrombin time, prothrombin time, and AP time with the hot water extract of SKK14 variety at a concentration of 5 mg/ml, the thrombin time was extended by 2.2 times compared to the non-added group, and the prothrombin time and AP time are all extended 15 times. On the other hand, the extracts of immature green apple and mature fruit of common apple did not show significant inhibition of thrombin, prothrombin and coagulation factors at the concentration of 5mg/ml, so anticoagulant activity was not recognized. In addition, as a result of evaluating the platelet aggregation inhibitory activity targeting the flower apple extract of the SKK14 variety, only the immature fruit extract was confirmed to have stronger platelet aggregation inhibitory activity than aspirin. The immature flower apple extract of the SKK14 variety did not show any hemolysis of human red blood cells. Therefore, it was confirmed that the SKK14 variety flower apple extract, especially immature fruit, can be developed as an antithrombotic agent that can replace aspirin, which has a high risk of side effects such as gastrointestinal disorders.

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

The active ingredient of the present invention can be used for the prevention or treatment of various diseases related to thrombosis. These diseases include, for example, arterial thrombosis, acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, blurred vision, epileptic seizures, pulmonary thrombosis , deep vein thrombosis, lower limb edema, pain and acute peripheral arterial occlusion, and the like, and as venous thrombosis, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous sinus thrombosis, and central retinal vein occlusion. In particular, the above-described specific active ingredient of the present invention can also be used as a blood coagulation inhibitor (anticoagulant) or platelet aggregation inhibitor (antiplatelet agent).

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

These pharmaceutical compositions may further include carriers, excipients, or diluents, and examples of suitable carriers, excipients, or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.

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

As a preferred embodiment, liquid preparations for oral use may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, Fragrance, preservatives, etc. may be included.

As a preferred embodiment, preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, 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. Conventional additives such as solubilizers, tonicity agents, suspending agents, emulsifiers, stabilizers, preservatives and the like may be included in the injection.

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 with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, 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 in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those 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 patient's age, sex, and weight, and is generally 1 to 5,000 mg per kg of body weight, preferably 100 to 3,000 mg per day. Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to 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. All modes 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 route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all common routes as long as it can reach the target tissue. can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering active ingredients to target cells.

In the present invention, "subject" includes, but is not particularly limited to, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. and, preferably, a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in a variety of ways, such as foods and beverages 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, etc., and can be used in the form of powders, granules, tablets, capsules or beverages. .

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

In addition to containing the above compound as an essential component in the indicated ratio, the health functional food of the present invention may contain food additives such as natural carbohydrates and various flavors as additional components.

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

As the flavoring agent, natural flavoring agents such as stevia such as thaumatin, rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The proportion 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 is various nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and enhancers, 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 fruit flesh for production of natural fruit juice, fruit juice beverages, and vegetable beverages. 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 one preferred embodiment 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 flower apple and general apple extract and analysis of components of the extract

Flower apples of the SKK14 variety cultivated and harvested at the Cheongsong-gun Agricultural Technology Center in Gyeongsangbuk-do in 2017 and ordinary apples of the Miamia Fuji variety were recovered 70 days after flowering and 130 days after flowering, respectively, to remove foreign substances, cut them, and extract hot water was prepared. Flower apples and general apple information used in the experiment are shown in Table 1 and FIG. SKK14 is used as a pollinator for late-flowering apple varieties (Fuji, Hwahong). The immature flower apple SKK14 used was 0.7cm in size, but the general green apple was 3.5cm, showing more than five times the size, and the mature flower apple SKK14 While it is 0.8 cm in size, mature common apples are 6.1 cm, showing about eight times the size.

[Table 1] Size and characteristics of flower apples and common apples

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

[Table 2] Physicochemical characteristics and color difference between flower apples and general apples

As shown in Table 2, in the case of flower apples, the increase in brix and acidity was insignificant as the moisture content decreased from 67.2% (70 days after flowering) to 60.6% (130 days after flowering) as they matured, but in the case of ordinary apples , as it matured, the water content increased from 80.4% (70 days after flowering) to 87.1% (130 days after flowering), and brix and acidity increased significantly. In addition, as a result of color difference analysis of the hot water extract, both flower apples and normal apples showed an increase in brightness and yellowness and a decrease in redness as they matured.

On the other hand, 20 times distilled water was added to flower apples and ordinary apples by weight, extracted once at 100 ° C. for 1 hour, the extracts were collected and filtered, and then concentrated under reduced pressure to prepare powder to prepare hot water extracts, respectively. The extraction efficiency and component analysis results of the extract are shown in Table 3. Component analysis determined total polyphenol, total flavonoid, total sugar and reducing sugar content. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of a saturated Na ₂ CO ₃ solution were added to 400 μl of the extraction sample solution, and the absorbance was measured at 725 nm after allowing to stand at room temperature for 1 hour. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was stirred and extracted with methanol for 18 hours, 4ml of 90% diethylene glycol was added to 400μl of the filtered extraction sample solution, 40μl of 1 N NaOH was added, and the absorbance was measured at 420nm after reacting at 37℃ for 1 hour. . Rutin was used as a standard reagent. Reducing sugar was quantified using the DNS method, and total sugar was quantified using the phenol-sulfuric acid method.

[Table 3] Extraction efficiency and useful component analysis of hot water extracts of flower apples and general apples

As shown in Table 3, the extraction efficiency of flower apples was lower than that of Fuji apples. In particular, in the case of ordinary apples, the extraction efficiency increased as they matured, whereas in the case of SKK14 flower apples, the extraction efficiency of mature fruits was lower than that of immature fruits. showed Therefore, in the case of SKK14, it is presumed that many flowers bloom and bear fruit at the beginning, and then, as they mature in a hard form without significant change, they finally drop naturally.

On the other hand, in the case of the SKK14 extract of immature flower apples, the polyphenol content was 150.0 mg/g, which was 2.3 to 11.7 times higher than that of mature flower apples, green apples, and mature apples. In addition, the flavonoid content of the SKK14 extract of immature flower apples was 2.7 to 9.1 times higher than that of mature flower apples, green apples, and mature apples. However, the total and reducing sugar contents were relatively low, and the acidity was about twice as high as that of apples. Therefore, it was confirmed that SKK14 of immature and mature flower apples has low functionality and is difficult to use for food, and it is desirable to develop it as a functional material.

Example 2: Blood coagulation inhibitory activity of flower apple and common apple extracts

The blood coagulation inhibitory activity of the flower apple and common apple extracts of Example 1 was evaluated, and the results are shown in Table 4. At this time, the blood coagulation 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; For plasma, commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and thrombin time, prothrombin time, and AP time measurements 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 the tube of Amelung coagulometer KC-1A (Japan), reacted for 2 minutes, and 100 μl of plasma was added. After that, the time until plasma coagulation was measured. As a control, aspirin (Sigma Co., USA) was used, and as a solvent control, DMSO was used instead of the sample. In the case of DMSO, the coagulation time was 24.2 seconds. The thrombin-inhibiting effect was expressed as the average of three or more repeated experiments, and the thrombin-inhibiting activity was expressed as the value obtained by dividing the coagulation time upon addition of the sample by the coagulation time of the solvent control.

prothrombin time

70μl of standard plasma (MD Pacific Co., China) and 10μl of sample solutions of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan), warmed at 37℃ for 3 minutes, 130μl of PT reagent was added and plasma coagulated The time until it was obtained was expressed as the average value of three repeated experiments. As a control, aspirin (Sigma Co., USA) was used, and as a solvent control, DMSO was used instead of the sample. In the case of DMSO, the coagulation time was 16.8 seconds. The prothrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time upon addition of the sample 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 warmed at 37°C for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added and the tube was incubated again at 37°C for 3 minutes. Incubated for minutes. 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 solidification time was 42.2 seconds. The result of aPTT was expressed as the average value of three repeated experiments, and the blood coagulation factor inhibitory activity was expressed as the value obtained by dividing the aPTT at the time of addition of the sample by the aPTT of the solvent control.

[Table 4] Blood coagulation inhibitory activity of flower apple and general apple extracts

As a result, as shown in Table 4, at a concentration of 5 mg/ml, the SKK14 variety of immature flower apple hot-water extract extended the thrombin time, prothrombin time, and AP time by more than 15 times compared to the non-additive group, and 2.5 mg/ml Even at the concentration, it was extended by 1.87, 3.69, and 1.17 times, respectively, indicating excellent thrombin, prothrombin, and coagulation factor inhibition compared to aspirin. In addition, as a result of measuring the thrombin time, prothrombin time, and AP time with the hot water extract of SKK14 variety at a concentration of 5 mg/ml, the thrombin time was extended by 2.2 times compared to the non-added group, and the prothrombin time and AP time are all extended 15 times. On the other hand, the extracts of immature and mature fruits of common apple did not show significant inhibition of thrombin, prothrombin and coagulation factors at the concentration of 5mg/ml, so anticoagulant activity was not recognized.

Example 3: Platelet aggregation inhibitory activity of flower apple and common apple extracts

The human platelet aggregation inhibitory activity of the SKK14 variety flower apple and common apple extracts of Example 1 was evaluated, and the results are shown in Table 5. Platelets are disc-shaped small cells that circulate in blood vessels along with various blood cells. They do not have nuclei, but instead have cytoplasmic granules containing high concentrations of various substances related to blood vessel damage protection and platelet aggregation. It is an important cell that secretes factors and binds to collagen exposed by damage to endothelial cells to form a primary hemostatic plug to initiate thrombogenesis. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombogenesis. 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 a 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, after re-suspending 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), at 3,000 rpm for 10 minutes After centrifugation, the cells were re-suspended in suspending buffer, and at this time, the platelet count was adjusted to be 4x10 ⁹ /ml. Then, 2.5 μl of 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 5] Platelet aggregation inhibitory activity of flower apple and general apple extracts

As shown in Table 5, at a concentration of 0.125 to 0.25 mg/ml, aspirin exhibited strong inhibition of platelet aggregation (aggregation degree of 35.3 to 49.2%) in a concentration-dependent manner, indicating evidence for its use as an antithrombotic agent in clinical practice. On the other hand, the hot-water extract of immature flower apples of the SKK14 variety strongly inhibited human platelet aggregation at a concentration of 0.25 mg/ml, showing better platelet aggregation inhibitory activity than aspirin. However, the hot-water extract of mature fruits promoted human platelet aggregation at a concentration of 0.25 mg/ml, resulting in a degree of aggregation of 138.3%. In the case of Fuji cultivar, which is a common apple, the concentration of 0.25 mg/ml had no effect on human platelet aggregation regardless of maturity. These results suggest that the immature flower apple extract of the SKK14 variety can be developed as an antiplatelet agent.

Example 4: Human erythrocyte hemolytic activity of flower apple extract

Flower apples have been processed and eaten as jam, vinegar, and liquor for a long time, and are currently registered as a food raw material in Korea, and their safety is secured. In the present invention, human erythrocyte hemolytic activity was evaluated to evaluate the acute toxicity of the flower apple extract, and the results are shown in Table 6. At this time, the hemolytic activity was evaluated according to the previous 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 was added to a 96-well microplate and various 100 μl of the sample solution was added and reacted at 37 ° C for 30 minutes. Then, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate. The degree of hemoglobin leakage due to hemolysis was measured at 414 nm did DMSO (2%) was used as a solvent control for the sample, and Triton X-100 (1 mg/ml) was used as a control for red blood cell hemolysis. Hemolytic activity was calculated using the following formula.

[Table 6] Human erythrocyte hemolytic activity of flower apple extract

As a result, as shown in Table 6, first, it was confirmed that DMSO and water used as controls had no hemolytic activity, and that triton X-100 haemolyzed 100% of red blood cells at a concentration of 1 mg/ml. On the other hand, flower apple and common apple extracts had no hemolytic activity at all regardless of maturity. Therefore, it is judged that the flower apple extract of the SKK14 variety will not present a separate problem of inducing acute toxicity.

Example 5: Plasma, acid and heat stability evaluation of SKK14 variety flower apple extract

Plasma stability, heat stability, and acid stability for antioxidant activity were confirmed for the flower apple extract of SKK14 variety obtained in Example 1 above. The above samples show a significant decrease in the antioxidant activity of DPPH anion scavenging ability, ABTS cation scavenging ability, and nitrite scavenging ability even when 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. showed high stability. Therefore, the extract is expected to maintain excellent antioxidant activity during digestion and absorption processes and food manufacturing processes.

Claims (4)

A platelet aggregation inhibitor containing SKK14 immature flower apple extract ( Malus prunifolia Wild. Borkh) as an active ingredient. The platelet aggregation inhibitor according to claim 1, wherein the extract is a hot water extract. delete delete

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