KR101800372B1 - Pharmaceutical composition comprising the extraction of unripe korean peaches 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 functional health food product for preventing or treating thrombosis, comprising an extract of unripe fruits of Prunus persica L. Batsch as an effective component. The extract of unripe fruits of Prunus persica L. Batsch, as an effective component of the pharmaceutical composition and the functional health food product of the present invention, shows potent antithrombotic activity through an inhibition effect on platelet aggregation which acts as an initiator of thrombus formation, along with an inhibition effect on thrombus formation-related enzymes and blood coagulation factors. Further, the extract of unripe fruits of Prunus persica L. Batsch shows no hemolytic activity to human red blood cells; has excellent thermal stability; and does not exhibit a decrease in inhibition activities on blood coagulation factors and thrombus formation-related enzymes even in an acid environment at pH 2 and in blood plasma, and thus may be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke through improving blood circulation. Furthermore, the effective component can be processed in various forms such as extracts, powder, pills, and tablets that can be conveniently ingested at all time, and thus are extremely useful in drug and food industries.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention or treatment of thrombosis, which comprises as an active ingredient, an extract of peach juice, and a health functional food. [0002] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis,

The present invention relates to a pharmaceutical composition and a health functional food for the prevention or treatment of thrombosis containing Prunus persica L. Batsch extract of unripe fruits as an active ingredient and more specifically, A pharmaceutical composition for prevention or treatment / improvement of thrombosis through inhibition of platelet aggregation inhibition and blood coagulation inhibition comprising hexane fraction or ethyl acetate fraction of hot water extract, ethyl acetate fraction, or butanol fraction and peach oil extract of hot water extract as an active ingredient And a health functional food.

As a constituent of human body, blood has various important functions such as oxygen and nutrients, the function and buffering function of waste products, maintenance of body temperature, control of osmotic pressure and maintenance of ion balance, maintenance of moisture, regulation of fluidity, maintenance and regulation of blood pressure, have. Normal blood circulation facilitates blood circulation by complementary regulation of the blood coagulation system and thrombolysis system in the body. Among them, the mechanism of the blood coagulation system is that the platelets adhere to the blood vessel walls and coagulate to form platelet thrombus , It is reported that the blood coagulation system is activated and fibrin thrombus is formed centering on the platelet aggregation mass.

On the other hand, the production of fibrin thrombus is activated by thrombin involved in fibrin clotting through several steps of a number of blood coagulation factors to finally produce a fibrin monomer from fibrinogen. The fibrin monomers are polymerized by calcium, Cells to form a cross-linked fibrin polymer by factor XIII and produce a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation by activating platelet, V factor, and Factor VII to promote blood coagulation. Therefore, the activity inhibitor of thrombin can be used as a prophylactic and therapeutic agent very useful for various thrombotic diseases caused by excessive blood coagulation. On the other hand, prothrombin activation after sequential activation of factor XII, factor XI, factor IX and factor X is known to activate thrombin in the endogenous thrombogenesis pathway, so that specific inhibition of blood coagulation factors is also important. It is becoming a target. To date, various anticoagulants such as heparin, coumarin, aspirin, and europaine have been used for the prevention and treatment of thrombotic diseases. However, these anticoagulants are not only very high in price, but also have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited.

On the other hand, peach ( Prunus persica L. Batsch) is a fruit of peach tree belonging to the rose and cherry tree and is a small arboreous tree which grows to 3 ~ 5m. It is also known as China 's copy - tree, and in Korea it is known to belong to the five fruit trees following apple trees, tangerine trees, persimmon trees and vines. In Korea, peaches are cultivated largely by Prunus persica for alba Schneider and Prunus persica for rubro-plena Schneider. Flesh is seasonal, including carbohydrates, organic acids, minerals, vitamins and enzymes. It plays a role of maintaining nutrition with fruits, and has a distinctive fruit flavor and is widely applied to various food industries. In Korea, peaches are mainly used for reproductive purposes, but they are poor in storage stability and difficult to store organs. In case of scratches, they are rapidly lost in commerciality and contain allergens such as allergens such as lipid transfer protein. Research on processed product development is essential. In fact, research on the development of processed foods such as peaches, juices, liquors, canned foods, etc. has recently been concentrated in Korea, and research on functional substances and efficacy of peaches is under way.

To date, research on peach flesh has focused on the characterization of fermented peaches (Jung, JH, et al., 2003. Korean Journal of Food Science and Nutrition 32: 506-512), Optimization of peach fermentation (Ki-dong, Korea Journal of Food Science and Nutrition 44: 586-591) , And antioxidant and anti-inflammatory effects of stem other than pulp (Cha Dae Cheon et al., 2004. Herbal Medicines 12: 289-294) and the whitening activity of flowers and calyxes (Korean Journal of Food Storage and Distribution, 19: 946-950). In addition, the effects of lipid components and blood pressure lowering in congenital hypertensive rats in relation to wild stone peach ( Prunus persica Batsch var. Davidiana Max.) (Kim, Hansoo, 2006. Life Science 16: 1071-1079), Streptozotocin- Effects of dietary lipid peroxidation on lipid peroxidation in diabetic rats (Han-Soo Kim, 2004. Korean Journal of Food Science and Nutrition 17: 337-345), and lipid peroxidation and creatine phosphokinase activity in diabetic rats 278), the effect of free fatty acids, creatine phosphokinase and LCAT activity on hypercholesterolemic rats (Kim, Hansoo, 2005. Korean Journal of Food Science and Nutrition 18: 265-271). In one room, peach blossoms, leaves, and seeds are used as herbal medicines rather than peaches, and they are known as dohwa, maple, and doin, and are known to help inflammation, hemostasis, eradication and blood circulation in women. In particular, mature peach seeds are known to have excellent effects on menstrual irregularity and menstrual occlusion. Pharmacological actions include vasodilation, increased blood flow, antiinflammatory, analgesic and antitumor effects (Kosuge T et al., 1985. Chemical Pharmaceutical Bulletin 33: 1496-1498). However, improvement of blood circulation and antithrombotically active substances in doenne are known as triolein (Kosuge T et al., 1985. Chemical Pharmaceutical Bulletin 33: 1496-1498) and other lipid components (Yang Nian-Yun, 2011. Natural Product Research 25: 1650-1656), there are restrictions on the use, and mature seeds contain toxic components of cyanide compounds such as amygdalin, and their removal is indispensable.

On the other hand, Peach Yukwa is a fruit produced in the enemy and the process that sprouts relatively small young fruit three to four times in order to raise peach, and 90% of the whole fruit belongs to it. It is a fact that it is being abolished.

The results of this study were as follows: 1) The analysis of the peach yuchu and the quality characteristics of domestic peach yuchu (Kim Da-Mi et al., 2012. J Korean Soc Food Sci Sci Nutr 41: 221-226) A study on the development of cosmetic materials using peach juice extracts (Kim, Dae-Mi et al., 2012. J Korean Soc Food Sci Sci Nutr 41: 156-160) Quality Characteristics of Peach Juice and Pickled Pickles during Storage (Hong, Min-Seo, et al., 2012. J Korean Soc Food Sci Sci Nutr 41: 1577-1583) . However, there is no known anti-thrombotic activity of mature pulp and peach juice until now.

On the other hand, patents relating to peaches include Korean Patent No. 10-0826311 entitled " Pharmaceutical composition containing peach extract having an inhibitory activity against hepatotoxicity and renal toxicity induced by anticancer drugs as an active ingredient ", Patent No. 10-1384377 No. 10-0863751 entitled " Functional Tobacco Filter Containing Peach Seed Activated Carbons ", " Patent No. 10-1051076 ", Composition for treating allergy comprising peach juice, And a composition for improving skin wrinkles containing an extract of peach blossom as an active ingredient, "and " Preparation method for skin wrinkles " Patent No. 10-0756669 discloses a " cosmetic composition containing a ceramic extract exhibiting whitening activity ". Also, as disclosed in Japanese Patent Application Laid-Open No. 10-2007-0041927, "a composition containing a green tea extract showing antioxidant, anti-cancer and whitening activity" However, no patent related to strong antithrombotic activity through inhibition of blood coagulation and inhibition of platelet aggregation in peach flesh and peach juice except peach seeds is known.

 Kosuge T, Ishida H, Ishii M. 1985. Studies on active substances in the herbs used for oketsu ("stagnant blood") in Chinese medicine. II. On the anticoagulative principle of persicae semen. Chemical Pharmaceutical Bulletin 33: 1496-1498.  Yang Nian-Yun, Liu Li, Tao Wei-Wei, Duan Jin-Ao, Liu Xun-Hong, Huang, Su-Ping. 2011. Antithrombotic lipids from Semen Persicae. Natural Product Research 25: 1650-1656.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and it is an object of the present invention to provide a pharmaceutical composition and a health functional food for preventing or treating thrombosis, I would like to.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis comprising an extract of peach juice extract as an active ingredient.

Preferably, the peach juice extract is a hot-water extract of peach juice.

Preferably, the hot-water extract is an ethyl acetate fraction or a butanol fraction obtained from a hot-water extract of peach oil.

Preferably, the peach juice extract is a hexane fraction or an ethyl acetate fraction obtained from an ethanol extract of peach yogurt.

Further, the present invention provides a health functional food for improving thrombosis comprising peach fruit body extract as an active ingredient.

The pharmaceutical composition for the prevention or treatment of thrombosis according to the present invention and the extract of peach juice as an active ingredient of the health functional food inhibit the coagulation inhibition effect of platelets which play a role of inhibiting thrombogenesis-related enzymes and blood coagulation factors, And exhibits excellent hemostatic activity against human erythrocytes, exhibits excellent thermal stability, and is in a condition of acidic pH 2 and loss of blood coagulation factor inhibitory effect and thrombogenic enzyme inhibitory effect even in plasma It is expected that it can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation and the active ingredient can be processed into various forms such as extract, powder, ring and tablet, Because it has excellent effects that can be prepared in the form of pharmaceuticals And a very useful invention in the food industry.

Fig. 1 is a photographic view of the peach fruit oil used in the present invention. Fig.
(DMSO), 2: aspirin (0.125 mg / ml), 3: aspirin (0.25 mg / ml), and the like. , 4: peach oil and hot water extract (0.25mg / ml), 5: peach oil and hot water extract (0.5mg / ml), 6: peach oil and hot water extract, hexane fraction (0.25 mg / ml), 8: peach oil and hot water extract butanol fraction (0.25 mg / ml), 9: water residue after organic solvent fractionation of peach oil and hot water extract (0.25 mg / ml).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of peach juice by the inventors of the present invention, peach fruit extracts were prepared by a certain method, and a hexane fraction, an ethyl acetate fraction, a butanol fraction and a water residue after an organic solvent fraction were used as an antithrombotic active ingredient The extracts and fractions were found to have excellent heat stability and acid stability without showing hemolytic activity against human erythrocytes. Thus, the extracts and fractions were used as pharmaceutical compositions for preventing or treating / improving thrombosis, Health functional foods.

Specifically, the inventors of the present invention conducted a study to develop a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis using peach yuwoo which is known to have excellent blood circulation improving effect in the private sector. Respectively. The extracts were subjected to sequential organic solvent fractionation with hexane, ethyl acetate, and butanol, respectively. Finally, the water residue was recovered after fractionation. The antithrombotic activity of each of the extracts and fractions was assessed by thrombin time, prothrombin time and activated partial thromboplastin time (aPTT) on human plasma and human thrombin As a result, the platelet aggregation inhibitory activity was excellent in the butanol fraction of the hot-water extract and the hot-water extract, the ethyl acetate fraction of the hot-water extract, the hexane fraction and the ethyl acetate fraction of the ethanol extract were found to have a strong blood coagulation inhibitory activity, Did not show hemolytic activity on human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis and a health functional food for preventing or ameliorating thrombosis, comprising an extract of peach juice as an active ingredient.

The peach juice refers to a peach having a weight of 5 g and a diameter of 5 cm or less without producing seeds.

In a preferred embodiment, the peach fruit extract can be a hot-water extract of peach juice, an ethyl acetate fraction or a butanol fraction of a hot-water extract.

In another preferred embodiment, the peach juice extract may be a hexene fraction or an ethyl acetate fraction obtained from an ethanol extract of peach oil.

Hereinafter, the peach juice extract and the production method and efficacy test of each active fraction material of the present invention will be described in more detail.

The present invention relates to a method for preparing an extract, comprising: preparing an extract from peach yuchu; Preparing sequential organic solvent fractions of hexane, ethyl acetate, and butanol from peach oak extract and preparing the resulting water residue; Evaluating the antithrombotic activity of the extract and the fractions and examining the stability of the active substance of the antithrombotic active fraction.

The "peach juice extract" contained in the composition of the present invention is obtained by extracting peach juice and washing and then cutting it, extracting it with an organic solvent, filtering the extract using a filter net of 0.06 mm or less, ≪ / RTI >

The organic solvent used in the present invention may be any organic solvent such as water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol (methanol, ethanol, alcohol, propanol, butanol etc.) having 1 to 4 carbon atoms, a mixed solvent of the lower alcohol and water , And hot water or 95% ethanol extraction is most preferred.

As a preferred embodiment, the peach oilseed extract of the present invention can be used by extracting peach oilseed with hot water or ethanol. Herein, the hydrothermal or ethanol extract may be sequentially or separately fractionated with an organic solvent of hexene, ethyl acetate and butanol to obtain a hexane fraction, an ethyl acetate fraction, a butanol fraction and a water residue.

In the present invention, when thrombin time, prothrombin time, apytime and human platelet aggregation inhibitory activity were measured at a concentration of 5 mg / ml of extract of peach juice, the increase in blood clotting time was insignificant as compared with no- The activity of platelet aggregation inhibitory activity was hardly recognized. The hot - water extract showed strong inhibition of platelet aggregation, whereas the ethanol extract showed platelet aggregation promoting effect.

However, in the case of the ethyl acetate fraction of peach oil and hot water extract, the concentration of thrombin time, prothrombin time and apitime time was prolonged in a concentration dependent manner. The hot water extract and the butanol fraction showed excellent human platelet aggregation inhibition at a concentration of 0.25 mg / ml. This suggests that peach oil and hydrothermal extract, ethylacetate fraction and butanol fraction thereof have a very strong antithrombotic activity and suggest that anticoagulants such as aspirin and antiplatelet agents, which have high risk of side effects, can be substituted.

In addition, the hexane fraction and the ethyl acetate fraction of the peach oil and ethanol extracts were confirmed to strongly inhibit blood coagulation-related enzymes by prolonging thrombin time, prothrombin time and apathy time in a concentration-dependent manner. In addition, the above extracts and fractions showed no hemolytic activity against human erythrocytes, and thus it was confirmed that the extracts and fractions were excellent in safety, ease of harvesting and economical efficiency as compared with the mature peach seeds.

The peach juice extract and the active fraction thereof of the present invention can be prepared into powders through a conventional powdering process such as vacuum drying and freeze drying or spray drying. They are not degraded by various degradation enzymes in the plasma, and maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The active ingredient of the present invention can be used for the prevention or treatment of various diseases associated with thrombosis. Such diseases include, for example, arterial thrombosis such as acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, , Deep vein thrombosis, lower limb edema, pain, and acute peripheral artery occlusion. Vein thrombosis includes deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the active ingredient of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, injections of sterilized injection solutions And may be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, 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, an antiseptic, and the like.

In a preferred embodiment, the solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, for example starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Perfumes, preservatives, and the like.

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may 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, "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 will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered 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, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

As a preferable example, 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 kg of body weight per day Or every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

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

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell.

In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , 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 for prevention or improvement of thrombosis. Examples of foods containing the active ingredient of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage .

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, and the health beverage composition may be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention may contain, as an additional ingredient, a food-acceptable food-aid additive such as natural carbohydrates and various flavors, in addition to containing the above-mentioned compound as an essential ingredient in the indicated ratio.

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

Examples of the flavoring agents include natural flavoring agents such as tautatin, rebaudioside A and stiglycerin such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. 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 may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such 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 by way of examples. The following examples are only exemplary embodiments of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

[Example]

Example 1 Preparation of Peach Extracts and Analysis of Their Useful Ingredients

From the peach trees cultivated in Cheongsong, Gyeongsang Province, the wheat was harvested from the peach trees (Elbert seeds, large and small seedlings, western seedlings and mixed seeds) weighing 5g or less (5cm in diameter), and washed with flowing water to remove foreign matter , Ethanol extracts and hot water extracts. The average moisture content of the peach juice was 2.31 ± 0.22cm, 2.57 ± 0.25cm, 3.98 ± 0.92g and 86.2 ± 0.45%, respectively. The pH of the peach juice was pH 4.32, brix 7.6 and acidity 0.62% (acetic acid equivalent) and the taste was so strong that it was difficult to reproduce. Table 1 shows the nutritional composition of peach yuwu as compared with that of mature peach. It shows higher contents of protein, fat and various minerals than mature peach, and especially rich in calcium, potassium and phosphorus contents . In addition, riboflavin, niacin, and vitamin C contents were relatively higher than those of maturity, so they were found to be nutritionally excellent although they were sensually problematic.

[Table 1] Comparison of Nutritional Components between Peach Yukwa and Maturity

In preparing the ethanol extract for the component analysis and activity evaluation, 10 times of ethanol was added to the peach oil and the samples were repeatedly extracted twice at room temperature, and the extracts were collected by filtration and concentrated under reduced pressure to prepare ethanol extracts . In the case of the hot-water extract, 10-fold of distilled water was added to the peach oil and samples, and the mixture was heated at 100 ° C for 1 hour and then cooled. The mixture was cooled again and the above- To prepare hot water extract. The results of each extraction efficiency and component analysis of the extract are shown in Table 2. Total polyphenols, total flavonoids, total sugars and reducing sugar contents were measured by compositional analysis. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau and 100 μl of saturated Na ₂ CO ₃ solution to 400 μl of the extract solution, leaving it at room temperature for 1 hour and measuring the absorbance at 725 nm. Tannic acid was used as a standard reagent. The total flavonoid content of each sample was measured by stirring for 18 hours in methanol. To the 400 μl of the filtered extract, 4 ml of 90% diethylene glycol was added, 40 μl of 1 N NaOH was added, and the absorbance at 420 nm was measured at 37 ° C. for 1 hour. As a standard reagent, rutin was used. Reducing sugar was determined by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 2] Extraction efficiency and useful components analysis of peach oil, ethanol extract and hot water extract

As shown in Table 2, peach juice showed 1.11 times higher extraction efficiency than ethanol extraction in hot water extraction. The total polyphenol contents of ethanol extracts and hot water extracts were 11.0 and 15.4 mg / g, respectively. The total content of flavonoids was 13.9 and 15.6 mg / g in hot water extract. Total sugar content of ethanol extract and hot water extract was 156.9 and 162.3 mg / g, respectively. In the case of reducing sugar, the ethanol extract had 56% of the total sugar content and the hot water extract had 76% of the total sugar content. The peach fruit extract contained a very high content of reducing sugar.

Example 2: Blood coagulation inhibitory activity of peach juice extract

The ethanol coagulation inhibitory activity of the ethanol extract and hot water extract of Example 1 was evaluated and the results are shown in Table 3. [ At this time, the evaluation method of blood clotting inhibitory activity of peach juice extract was evaluated according to the previously reported method (Sohn et al., 2004. Kor J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14: 509-513; Ryu et al. 2010. J. Life Science, 20: 922-928), thrombin time, prothrombin time and apathy time were measured. Plasma was obtained from a commercial control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and the thrombin time, prothrombin time and api assay were performed as follows.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA) and 50 μl of 20 mM CaCl ₂ and 10 μl of various concentrations of sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C., and 100 μl of plasma was added And the time until the plasma coagulated was measured. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 24.2 seconds. The thrombin inhibitory effect was expressed as the average value of the experiments repeated three or more times. The thrombin inhibitory activity was expressed by the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

Prothrombin time

Add 70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution to tubes of Amelung coagulometer KC-1A (Japan), heat at 37 ° C for 3 minutes, add 130 μl of PT reagent, The time from the start of the experiment to the start of the experiment was expressed as the average value of the experiments repeated three times. As a control, aspirin (Sigma Co., USA) was used and DMSO was used as a solvent control instead of the sample. DMSO showed a clotting time of 16.8 seconds. The prothrombin inhibitory activity was expressed as the value obtained by dividing the solidification time at the time of addition of the sample by the solidification time of the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of various concentrations of sample extract were added to a tube of Amelung coagulometer KC-1A (Japan), and the mixture was heated at 37 ° C for 3 minutes. Then 50 μl of aPTT reagent (Sigma, ALEXINTM) Lt; / RTI > After the addition of 50 μl CaCl ₂ (35 mM), the time until the plasma coagulated was measured. As the solvent control, DMSO was used instead of the sample. In this case, the solidification time was 42.2 seconds. The results of aPTT were expressed as the mean value of three repeated experiments, and the coagulation factor inhibitory activity was expressed as aPTT time divided by the aPTT time of the solvent control.

[Table 3] Blood coagulation inhibitory activity of peach oil, ethanol extract and hot water extract

As a result, the extracts of peach juice showed very little blood coagulation inhibitory activity at the concentration of 5 mg / ml, and the ethanol extract showed a slight prolongation of 1.24 times thrombin time and showed thrombin inhibitory activity. At this time, aspirin used as a control was lengthened by 1.72 times, 1.31 times, and 1.3 times, respectively, at thrombin time, prothrombin time, and apathy time at a concentration of 1.5 mg / ml.

Example 3: Platelet aggregation inhibitory activity of peach juice extract

The human platelet aggregation inhibitory activity of the ethanol extract and hot-water extract of Example 1 was evaluated, and the results are shown in Table 4. Platelets are cytoplasmic granules that contain various substances related to blood vessel damage protection and platelet aggregation at high concentration instead of nucleus, and circulating blood vessels together with various blood cells. It is an important cell that secretes factors and binds to collagen exposed by damage of endothelial cells to form a primary hemostatic plug to initiate thrombogenesis. Therefore, inhibition of platelet aggregation is a very important activity to prevent thrombogenesis. Platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity (Platelet aggregation inhibition activity)

Human platelets were used as platelets and 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, the cells were resuspended in a suspending buffer (138 mM NaCl, 2.7 mM KCl, 12 mM NaHCO ₃ , 0.36 mM NaH ₂ PO ₄ , 5.5 mM Glucose, 0.49 mM MgCl ₂ , 0.25% gelatin, pH 7.4) and incubated at 3,000 rpm for 10 minutes After centrifugation, the cells were resuspended in a suspending buffer and the platelet count was adjusted to 4 × ^{10 9} / ml. Then platelet aggregation was measured at 37 ° C using a whole-blood agarometer (Chrono-log, USA) after 2.5 μl of collagen was added to 1 ml of suspension and reacted for 5 minutes.

[Table 4] Platelet Aggregation Inhibitory Activity of Peach Oil, Ethanol Extract and Hot Water Extract

 As shown in Table 4, first, aspirin showed strong inhibition of platelet aggregation in a concentration dependent manner at a concentration of 0.125 to 0.25 mg / ml, and thus it was found that the aspirin was used as an antithrombotic agent in clinical practice. Peach oil and hydrothermal extract showed excellent human platelet aggregation inhibitory activity at a concentration of 0.25 mg / ml. However, the peach oil and ethanol extract showed a strong platelet aggregation promoting activity at a concentration of 0.25 mg / ml.

Example 4 Preparation of Sequential Organic Solvent Fractions of Peach Oil and Hot Water Extract and Their Component Analysis

The peach oil and hot water extracts obtained in Example 1 were prepared in large quantities, and then subjected to sequential organic solvent fractionation with hexane, ethyl acetate and butanol. Finally, the water residue was recovered. The extraction efficiency, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction are shown in Table 5.

[Table 5] Comparison of yield and composition analysis of peach oil and hot water extracts and their sequential fractions

As shown in Table 5, the peach oil and hot water extracts were largely converted to butanol fractions and water residues, and the hexane fraction and the ethyl acetate fraction were 0.02 and 1.74%, respectively. This result means that about 0.11 g of the ethyl acetate fraction and about 0.58 g of the butanol fraction can be recovered from 100 g of the peach yoghurt which has been dehydrated. On the other hand, total polyphenol contents were 4.6 times and 2.26 times higher in ethyl acetate fraction and butanol fraction than peach oil and hot water extract, respectively, and 2.3 times and 1.99 times higher in total flavonoid content, respectively. Total sugar and reducing sugar analyzes showed higher values in the butanol fraction than in the water residue after the organic solvent fraction. Therefore, ethyl acetate fraction and butanol fraction of peach oil extract were expected to exhibit various physiological activities with high content of polyphenols and flavonoids. As a result of the actual analysis, the fractions showed strong antioxidative activity and nitrite scavenging ability.

Example 5 Evaluation of Blood Coagulation Inhibitory Activity of Sequential Organic Solvent Fractions of Peach Oil and Hot Water Extracts

The blood coagulation inhibitory activity of the peach oil and hydrothermal extract and the fractions thereof obtained in Example 4 was evaluated in the same manner as in Example 2, and the results are shown in Table 6.

[Table 6] Anticoagulant activity of peach oil and hot water extract and its fractions

As shown in Table 6, when aspirin was eluted at a concentration of 1.5 mg / ml, prolonged thrombin time, prothrombin time, and apathy time were increased by 1.72 times, 1.31 times, and 1.30 times, respectively. Among the fractions of the peach juice extract, the ethyl acetate fraction showed an excellent blood coagulation inhibitory activity by prolonging the thrombin time, the prothrombin time and the apathy time by 2.17 times, 1.51 times and 1.57 times at the concentration of 5 mg / ml, As the treatment concentration increased, the inhibitory activity increased. These results suggest that the ethyl acetate fraction of peach juice effectively inhibits thrombosis caused by intrinsic and extrinsic pathways and could be developed as a commercial antithrombotic agent. In addition, aspirin, which is a conventional antithrombotic agent showing side effects such as gastrointestinal disorders, It is possible to do. In addition, the fractions of peach juice are expected to exhibit excellent antioxidative and carcinostatic activities, thereby contributing to blood circulation improvement and antithrombotic activity additionally.

Example 6 Evaluation of Human Platelet Aggregation Inhibitory Activity of Sequential Organic Solvent Fractions of Peach Oil and Hot Water Extracts

The human platelet aggregation inhibitory activity of the peach oil and hydrothermal extract and the fractions thereof obtained in Example 4 was evaluated in the same manner as in Example 3, and the results are shown in Table 7 and FIG.

[Table 7] Human Platelet Aggregation Inhibitory Activity of Peach Oil and Hot Water Extract and Their Fractions

As shown in Table 7 and FIG. 2, the hot-water extract of peach yuangwu showed strong platelet aggregation inhibitory activity, and the fractions thereof showed strong coagulation inhibitory activity in the butanol fraction.

Example 7 Preparation of Sequential Organic Solvent Fractions of Peach Oil and Ethanol Extract and Analysis of Their Components

The peach oil and ethanol extracts obtained from Example 1 were prepared in large quantities, and then subjected to sequential organic solvent fractionation with hexane, ethyl acetate and butanol. Finally, the water residue was recovered. The extraction efficiency, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction are shown in Table 8.

[Table 8] Comparative analysis of yield and components of peach oil and ethanol extracts and their sequential fractions

As shown in Table 8, the peach oil and ethanol extracts were largely transferred to the butanol fraction and the water residue after fractionation, and the hexane fraction and the ethyl acetate fraction were also changed to 8.42 and 3.97%, respectively. This result means that about 0.49 g and 0.23 g of the hexane fraction and the ethyl acetate fraction can be recovered from 100 g of peanut buttermilk with the foreign substance removed. Ethyl acetate fraction, ethyl acetate fraction, butane fraction, hexane fraction and water residue were higher than ethyl acetate fraction in total polyphenol contents. Ethyl acetate fraction, ethyl acetate fraction, The most active components of the peach oil and ethanol extracts appeared to be the fat soluble ethyl acetate fraction. On the other hand, total sugar and reducing sugar analysis showed high contents in water residue and butanol fraction and unusually high total sugar content in ethyl acetate fraction. Therefore, the ethyl acetate fraction and the hexene fraction of the peach oil extract were expected to show various physiological activities. As a result of the analysis, the fractions showed strong antioxidative activity and nitrite scavenging ability.

Example 8 Evaluation of Blood Coagulation Inhibitory Activity of Sequential Organic Solvent Fractions of Peach Oil and Ethanol Extracts

The blood coagulation inhibitory activity of the peach oil and ethanol extract and the fractions thereof obtained in Example 7 was evaluated in the same manner as in Example 2, and the results are shown in Table 9. [

[Table 9] Anticoagulant activity of peach oil, ethanol extract and its fractions

As shown in Table 9, the hexane fraction and the ethyl acetate fraction of the ethanol extract fraction of peach yoghurt showed better thrombin, prothrombin, and coagulation factor inhibitory activity than the antithrombotic effect at the concentration of aspirin 1.5 mg / ml at the concentration of 5 mg / ml The inhibitory activity was increased with increasing treatment concentration. Especially, at the concentration of 7mg / ml of ethyl acetate fraction, thrombin time and prothrombin time were increased by 15 times or more as compared with the non - supplemented group, showing strong anticoagulant activity. These results suggest that hexane and ethylacetate fractions of ethanol extract of peach yoghurt can be developed as a commercial antithrombotic agent and replace aspirin, which is a conventional anti - thrombotic agent showing side effects such as gastrointestinal disorders. In case of peach juice, it is not necessary to remove toxic substances such as amygdalin because it is not produced in seeds. It is 90% of peach fruit and it is not used for other purposes. Therefore, it is very economical to improve blood circulation and develop antithrombotic agent I think it is possible.

Example 9: Human erythrocyte hemolytic activity of peach juice extract and its fractions

Peach juice is known to be free from allergies and toxic substances unlike maturity, and recently it has been developed and edible as a pickle (Hong Min, Seo et al., 2012. J Korean Soc Food Sci Nutr 41: 1577-1583; Master Thesis). To evaluate the possibility of acute toxicity of various extracts and their fractions of peach juice, human erythrocyte hemolytic activity was evaluated, and the results are shown in Table 10. The hemolytic activity was assessed in accordance with the existing reports (Jung In-chang, Son Ho Yong, 2014 ㆍ Korean J. Microbiol. Biotechnol. 42: 285-292). In brief, 100 μl of human erythrocytes washed three times with PBS were diluted in 96-well microplates , 100 μl of various concentrations of sample solution was added and the reaction was allowed to proceed at 37 ° C for 30 minutes. Then, the reaction solution was centrifuged for 10 minutes at 1,500 rpm, and 100 μl of the supernatant was transferred to a new microtiter plate. The hemoglobin efflux 414 nm. Triton X-100 (1 mg / ml) was used as an experimental control for erythrocyte hemolysis. DMSO (2%) was used as a solvent control of the sample. Hemolytic activity was calculated using the following formula.

[Table 10] Human erythrocyte hemolytic activity of peach juice extract and its fractions

First, DMSO and water used as controls did not have hemolytic activity, and triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. On the other hand, at the concentration of 0.5 mg / ml, the water residue of the ethanol extract and hot water extract showed slight hemolysis activity of 6.1 and 10.8%, respectively. However, Emphoteracin B, which is a known hemolytic agent, has a concentration of 0.0125 mg / ml , The hemolytic activity of these compounds is considered to be negligible. On the other hand, except for the extracts of peach juice and fractions thereof, it was confirmed that there was no erythrocyte hemolysis phenomenon up to a concentration of 0.5 mg / ml, so that there was no acute toxicity and erythrocyte hemolysis activity.

Example 10: Evaluation of Plasma, Acid and Thermal Stability of Ethyl Acetate Fraction, Butanol Fraction and Ethanol Extract of Hexane Fraction and Ethyl Acetate Fraction of Peach Oil and Hot Water Extract, Hot Water Extract, Ethyl Acetate Fraction, Butanol Fraction and Ethanol Extract

The hexane fraction or the ethyl acetate fraction of the peach oil and hot water extracts, the ethyl acetate fraction or the butanol fraction and the peach oilseed fraction of the hot-water extract obtained in Examples 1, 4 and 7 was used to measure plasma stability, thermal stability and anti- Acid stability was confirmed. The 5 samples did not show any significant inhibition of blood clotting and platelet aggregation inhibition activity even after 1 hour heat treatment at 100 ° C, 1 hour treatment with pH 2 (0.01M HCl), 1 hour treatment with plasma, Respectively. Thus, the extracts and active fractions described above are expected to maintain antithrombotic activity during digestion and absorption processes and food production.

Claims (5)

A pharmaceutical composition for inhibiting blood coagulation comprising an ethyl acetate fraction of an extract of peach juice as an active ingredient. [Claim 7] The pharmaceutical composition according to claim 1, wherein the peach juice extract is a hot-water extract or an ethanol extract of peach juice. delete delete delete

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