KR101725623B1

KR101725623B1 - Pharmaceutical composition comprising the organic solvent fraction of sorbus commixta fruit as an effective component for prevention or treatment of thrombosis and health functional food comprising the same

Info

Publication number: KR101725623B1
Application number: KR1020150038682A
Authority: KR
Inventors: 손호용; 김순화; 김미선
Original assignee: 안동대학교 산학협력단
Priority date: 2015-03-20
Filing date: 2015-03-20
Publication date: 2017-04-12
Also published as: KR20160113435A

Abstract

The present invention relates to a process for preparing Sorbus commixta ) fruit or an organic solvent fraction obtained from an ethanol extract as an active ingredient, and to a health functional food and a pharmaceutical composition for prevention / treatment / improvement of thrombosis. The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the hexane fraction and the ethyl acetate fraction of the Aspergillus oryzae as an active ingredient of the health functional food can be obtained by dissolving the beans in hot water or ethanol The hexane fraction and the ethyl acetate fraction are prepared by extracting and preparing a hexane fraction and an ethyl acetate fraction obtained by successively fractionating with hexene, ethyl acetate and butanol. The hexane fraction and the ethyl acetate fraction as described above are used for the production of a thrombogenic enzyme And exhibits potent antithrombotic activity by inhibiting the coagulation of platelets which plays a role of initiating thrombogenesis together with inhibition of blood coagulation factors and can be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation An outstanding efficacy A. Particularly, the hexane or ethyl acetate fraction of the extract of Aspergillus oryzae according to the present invention is excellent in thermal stability and does not exhibit the effect of inhibiting blood coagulation factor and thrombogenesis-related enzyme even in an acidic condition of pH 2 and plasma, Powder, ring, tablet, etc., and can be prepared in a form that can be taken at any time. Therefore, it is an extremely useful invention in the pharmaceutical industry and the food industry.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing an organic solvent fraction of an extract of Fusarium exarbibata as an effective ingredient. BACKGROUND ART FOOD COMPRISING THE SAME}

The present invention relates to a process for preparing Sorbus commixta ) fruit or an organic solvent fraction obtained from an ethanol extract as an active ingredient, and to a health functional food and a pharmaceutical composition for prevention / treatment / improvement of thrombosis.

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 , The blood coagulation system is activated and fibrin thrombi are formed centering on 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.

Sorbus commixta is a deciduous arboreous tree of the rosewood rosaceae. It is also called mountain ash, or mountain ash, in the West, and is a tree in which sprout-like buds emerge in spring. In Korea, it grows well everywhere in the country, but there is a large native colony especially in Chonnam, Gangwon and Ulleungdo. The height of the tree ranges from 6 to 8 meters. Flowers bloom in May to June, and the fruit ripens in red from September to October. In Korea, in addition to rowan, amurensis ) and Sagus sambucifolia ), and so far it has not yet clearly defined the origin of the plant, and the oriental herb has been used without any discrimination.

Rake is used for edible use of fruit and stem bark, and the stem is available for limited use as a food source. In one room, the fruit is called as maga, and it is picked up and dried and dried in the sun. It is known to suppress neuralgia. 1-sorbose, sorbic acid, and alpha-carotene are known to be the major components of margarine (Kim, Chang Eun et al. 1972. Journal of the Korean Society of Food Science and Technology) , 4: 1 ~ 5), diuretic, Jinhae, genome, Tangjang, and igal. In Europe, the production of liquor using the fruit of the rowan is common, and in particular, the distillate of the rowan fermentation is sold as an expensive product (Jo, HC et al., Journal of the Korean Society of Food Science and Nutrition, 2013. 42: 743 ~ 752).

On the other hand, the bark of the legs has been used as a medicine to protect the kidneys by being called mahi (馬牙皮) or hole blooms, and is used for bronchitis, rheumatoid arthritis, paralysis, gastritis and ossicles. In the case of leaves of Rokaba, it is not designated as a raw material for food, so it is not edible.

In the present study, we investigated the antioxidative activities of the extracts of R. japonica extracts on the lipid metabolism in rats (Jung, Byeonghee et al., 2003. 11: 143 ~ 147) (Korean Journal of Cosmetic Scientists, 2014. 40: 45 ~ 54), antioxidative activity of the oriental herb and other herbal medicinal compounds Inhibition of Angiotensin Converting Enzyme Inhibitory Effect of Methanol Extracts from the Rice Paddy Rice Extract (Jung Eun Jung et al., 2010. 11: 3358 ~ 3365) (2002), "Antioxidant and whitening of wisteria japonica", Wrinkle improvement study (Kyungnam et al., 2011. Journal of the Korean Chemical Society, 28: 482 ~ 490) 18: 282 ~ 288; Kang Mi-ae et al., 201 (Kang et al., 2007, Am J J Anaesthesiol. 2007; Arch Pharm Res., 30: 1116 ~ 1123), inhibition of vascular inflammation (Chol. Med., 35: 265 ~ 277) and anti-arteriosclerotic effect (Sohn et al., 2005. Biol. Pharm. Bul.l, 28: 1444 ~ 1449) Have not been reported.

In addition, the main registered patents related to Rake are Korean Patent No. 10-1265955 entitled " Cosmetic Composition Comprising Microcapsules Incorporated with Fermented Extract Mixed Herb Medicine ", No. 10-1076482 "Smoking with Rake Extracts as Active Ingredient (SG-II) for inhibiting osteoclast differentiation inhibition or chondrocyte differentiation promotion, which comprises a locust and a locally super extract, " No. 10-0247130 " A method of manufacturing a beverage containing bean as a main component and a beverage for preventive and joint treatment beverages, and a method of manufacturing a bean curd extract having an increased content of flavonoids and a cosmetic composition containing the same are disclosed in Japanese Patent No. 10-0697319. There are various registered patents relating to the manufacture of beverages and tea using bean sprouts. In the 10-0412785, "a tea composition for improving blood circulation and its preparation method ", and 10-0388080, Natural tea and its production method ", No. 10-0381389, "Method for manufacturing tea using artichoke fruit," No. 10-0516889, " &Quot; and " a method for producing the same ". Also, in the 10-0497799, a "method for producing distilled spruce branches" is also known. Recently, a cosmetic composition for alleviating skin irritation and alleviating skin inflammation (Korean Patent Laid-open No. 10-2014-0071738, June 12, 2014) and a composition for slimming cosmetic (Korean Patent Laid- 2014-0032645, March 17, 2014) are also disclosed. However, the pharmaceutical composition for prevention or treatment of thrombosis using the potent blood coagulation inhibiting effect of the rowan as far as the patent for the health functional food is not known.

KR 10-0412785 B

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 method for treating a thrombotic disease comprising a specific organic solvent fraction obtained from hot water or ethanol- A prophylactic or therapeutic pharmaceutical composition, a blood coagulation inhibitor, a platelet aggregation inhibitor and a health functional food.

In order to solve the problems as described above, the present invention Rowan (Sorbus commixta ) fruit was extracted from the group consisting of the ethyl acetate fraction of the hot-water extract, the hexane fraction of the ethanol extract and the ethyl acetate fraction of the ethanol extract, which were obtained by sequentially fractionating the hot water or ethanol extract of the fruit with an organic solvent of hexane, ethyl acetate and butanol, Or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention relates to a process for preparing Sorbus commixta ) fruit, the ethyl acetate fraction of the hot-water extract, the ethyl acetate fraction of the ethanol extract, and the mixture thereof, obtained by successively fractionating the hot water or the ethanol extract with an organic solvent of hexane, ethyl acetate and butanol, And a blood coagulation inhibitor comprising as an active ingredient.

In addition, the present invention relates to a process for preparing Sorbus commixta ) fruit of the present invention is selected from the group consisting of hexane fraction, ethyl acetate fraction and mixtures thereof obtained by successively fractionating an ethanol extract of the fruit with an organic solvent of hexene, ethyl acetate and butanol, as an active ingredient. do.

In addition, the present invention relates to a process for preparing Sorbus commixta ) fruit was extracted from the group consisting of the ethyl acetate fraction of the hot-water extract, the hexane fraction of the ethanol extract and the ethyl acetate fraction of the ethanol extract, which were obtained by sequentially fractionating the hot water or ethanol extract of the fruit with an organic solvent of hexane, ethyl acetate and butanol, Which is selected from the group consisting of at least one kind of active ingredient, as an active ingredient.

The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the hexane fraction and the ethyl acetate fraction of the Aspergillus oryzae as an active ingredient of the health functional food can be obtained by dissolving the beans in hot water or ethanol The hexane fraction and the ethyl acetate fraction are prepared by extracting and preparing hexane fraction and ethyl acetate fraction obtained by sequentially fractionating the mixture using hexane, ethyl acetate and butanol. Exhibits potent antithrombotic activity by inhibiting the coagulation of platelets which plays a role of initiating thrombogenesis together with inhibition of enzyme and blood coagulation factors and is used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation Can jump I have an effect. In particular, the hexene and / or ethyl acetate fractions of the extract of Fusarium exantans of the present invention are excellent in thermal stability and do not show loss of blood coagulation factor inhibitory effect and thrombogenesis-related enzyme inhibitory effect even in an acidic condition of pH 2 and plasma, It is an extremely useful invention in the pharmaceutical industry and the food industry because it can be prepared into various forms such as extract, powder, ring, and tablet and can be prepared at any time.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention recovered the antithrombotic active ingredient from the hexane fraction and the ethyl acetate fraction of the Aspergillus oryzae extract obtained by a certain method in order to test antithrombogenic activity against Sorbus commixta , The present invention has been made to utilize the extract as a pharmaceutical composition and health functional food for prevention or treatment / improvement of thrombosis by confirming that it has no hemolytic activity and is excellent in thermal stability and acid stability.

Specifically, the inventors of the present invention prepared a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis using an allergen, which is known to have excellent blood improving effect in a private sector, Thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (aPTT) for human plasma and human thrombin, respectively, , And it was confirmed that antitumor activity was only found in the bark of the rice bran, and the antithrombotic activity could not be confirmed in the fruit and leaf extracts themselves.

However, the ethanol extract or hot-water extract of Rice bran extract was sequentially fractionated in an organic solvent to obtain hexane fraction, ethyl acetate fraction, butanol fraction and water residue, and the above antithrombotic activity was reevaluated. As a result, Acetate fractions showed strong thrombin and prothrombin inhibitory activity and specific inhibitory activity of blood coagulation factors. In addition, the activity of inhibiting human platelet aggregation in the ethylacetate fractions of ethanol extracts was confirmed. In addition, the hexane fraction of the ethanol extract showed strong inhibitory activity on human platelet aggregation, and thus it was confirmed that the antitumor agent was highly developed. These fractions do not show acute oral toxicity because they do not show hemolysis of human erythrocyte, and no loss of activity occurs even at acid treatment at pH 2, heat treatment at 100 ° C, and plasma treatment, thus completing the present invention.

In order to confirm such an effect, the inventors of the present invention prepared extracts of R. melanogaster and sequential organic solvent fractions thereof, analyzed useful components such as total polyphenols, total flavonoids and the like, The prolongation of the active thromboplastin time and inhibition of human platelet aggregation by the inhibition effect, the prothrombin inhibitory effect, the inhibition of the blood coagulation factors (Factor VIII, Factor IX, Factor XI and Factor XII) And / or the ethyl acetate fraction has an antithrombotic activity superior to that of aspirin (trade name: Protect), which is a commercially available antithrombotic agent, and does not exhibit human erythropoietic activity, and the plasma, heat and acid stability And it was confirmed that the food processing suitability and stability were excellent.

Therefore, the present invention relates to a method for producing Sorbus commixta ) fruit was extracted from the group consisting of the ethyl acetate fraction of the hot-water extract, the hexane fraction of the ethanol extract and the ethyl acetate fraction of the ethanol extract, which were obtained by sequentially fractionating the hot water or ethanol extract of the fruit with an organic solvent of hexane, ethyl acetate and butanol, Or a pharmaceutically acceptable salt thereof as an active ingredient.

In a preferred embodiment, each of the fractions can be extracted with various solvents including water or ethanol. Preferably, the fractions are obtained from a hot-water extract or an ethanol extract. The active fractions include a hexane fraction having a strong platelet aggregation inhibitory activity, And the platelet aggregation inhibiting activity, or a mixture of two or more of these fractions.

Hereinafter, the production methods and efficacy tests of the respective fraction components produced from the Fagus exothermic extract of the present invention will be described in more detail.

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

The "Raspberry fruit extract" contained in the composition of the present invention is prepared by collecting mature fruit of September to October, washing, pulverizing, extracting with an organic solvent, and filtering the extract with a filter net of 0.06 mm or less Followed by filtration and concentration under reduced pressure. 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.

In the present invention, thrombin time, prothrombin time, and apitime time were measured at a concentration of 5 mg / ml of hot water or ethanol extract of Rana cunea fruit, and there was almost no change compared to no-addition catechins. However, the ethyl acetate fraction of the ethanol extract As a result of measuring thrombin time, prothrombin time, and apitime time after the addition, the coagulation time was prolonged more than 15 times, respectively, as compared with the non-addition fraction. Thus, the ethyl acetate fraction of the ethanol extract showed a very strong anti- . This strong coagulation inhibitory activity was also observed in the ethyl acetate fraction of the hot water extract. In the case of aspirin (trade name: Protect), which is a commercially available anti-thrombotic agent, considering that the thrombin time, prothrombin time and apathy time are extended 1.8 times, 1.9 times and 1.9 times, respectively, Ethylacetate fractions of Ricinus scavengers or ethanol extracts suggest that anticoagulants such as aspirin, which are highly likely to cause side effects, can be substituted. As a result of evaluation of platelet aggregation inhibitory activity using human platelets, the ethanol extract and hot water extracts of R. melanogaster did not exhibit platelet aggregation inhibitory activity. However, the hexane fraction and the ethyl acetate fraction of the ethanol extract were more than two times stronger than aspirin The hexane fraction, ethylacetate fraction and mixtures thereof of the ethanol extract of Rocaburi fruit were confirmed to be useful as platelet aggregation inhibitors.

The hexane fraction and the ethyl acetate fraction of the Rectum fruit extract of the present invention may be powdered by a conventional powdering process such as vacuum distillation, freeze drying, spray drying and the like. 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 fractions of the Rectal Fruit of the present invention may 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.

In a preferred embodiment of the present invention, the effective amount of the active fraction of Fusobacterium acanthomex extract in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and is generally 1 to 5,000 mg, 3,000 mg may be administered daily or every other day or one or 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. The food containing the active fractions of the extract of Fusobacterium maximus L. extract of the present invention includes various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and is in the form of powders, granules, tablets, capsules or beverages .

The active fraction of the extract of Fagopyrum rugosum according to the present invention may be added in an amount of 0.01 to 15% by weight based on the total weight of the whole food, and the health beverage composition may be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, .

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 agent include tau martin; Natural flavoring agents such as stevia such as rebaudioside A or 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 proportion of such an additive is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the organic solvent fraction of the extract of the legume fruit of the present invention.

Hereinafter, the present invention will be described in more detail with reference to 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 One: rowan Fruit extract and sequential organic solvent Fraction Preparation and analysis of their components

In July 2014, the immature fruit and the mature fruit of September are purchased from Ulleungdo, and 10 times of distilled water is added to each sample. The mixture is heated at 100 ℃ for 1 hour and then cooled. And the mixture was filtered and concentrated under reduced pressure to prepare a hot water extract. The extraction efficiency of each part, the pH of the extract, and the brix are shown in Table 1. The total polyphenol, total flavonoid, total sugar and reducing sugar content were determined by analyzing the components of each extract. 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 quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

[Table 1] rowan The extraction efficiency of the fruit extract and the pH , brix And component analysis comparison

As shown in Table 1, extraction efficiency was 2.5 times higher than that of immature fruit, and the pH was 5.0 in adult fruit and 4.2 in mature fruit. The brix of the extract was 3 times higher than that of immature fruit. On the other hand, the total polyphenol content and total flavonoid content were 4.4 times and 1.96 times higher than that of mature fruit, respectively. Especially, total sugar and reducing sugar content were 3.0 times and 2.1 times Respectively. Therefore, it is presumed that the mature fruit has a less sour taste than the immature fruit, and contains a large amount of organic acid of sour taste. Lastly, mature fruit was used for future experiments, considering economical efficiency, ease of harvesting and extraction efficiency.

In order to prepare the fractions of Fusarium oxysporum extract, first, hot water extracts and ethanol extracts were prepared using mature Fusarium fruit harvested in September, 2014. The hot-water extract was prepared in the same manner as above. 30 L of water was added to 3 kg of bean curd and the mixture was heated at 100 ° C. for 1 hour. The extract was collected by filtering three times, and then concentrated under reduced pressure to obtain powder. The ethanol extract was prepared by adding 30 L of 95% ethanol to 3 kg of beans and then extracting it three times at room temperature for 24 hours. The extract was collected by filtration and concentrated under reduced pressure to give powder. Table 2 shows the extraction efficiency, and the results of sequential organic solvent fractionation and component analysis of the extracts are shown in Table 3.

[Table 2] rowan Mature fruit Heat number And ethanol extract

In the case of mature fruit, the final extraction efficiency was higher than that of ethanol extraction, but the difference was not as high as 3%. Also, it was confirmed that three times repeated extraction was sufficient for each solvent.

[Table 3] rowan Mature fruit Heat number And ethanol extracts Fraction Comparison of water component analysis

In the case of the hot water extract of fruit, 81.1% was fractionated as water residue and most of it was water-soluble substance. In the case of ethanol extract, the fraction of hexane was 3.2% and the content of water residue was 68.4%. The hot - water extracts and the ethyl acetate fractions of the extracts of Ricinus japonica containing very high concentrations of polyphenols and flavonoids accounted for 1.1% and 2.1% of the extracts, respectively. This means that 0.243 g of the ethyl acetate fraction of the hot-water extract and 0.4 g of the ethyl acetate fraction of the ethanol extract can be recovered from 100 g of the sorghum extract. Ethylacetate fraction of Fusarium oxysporum showed strong antioxidant activity, which is expected to contribute to blood circulation improvement and aging suppression.

Example 2: rowan Fruit extract and sequential organic solvent Fraction Evaluation of blood coagulation inhibitory activity

Table 4 shows the results of evaluating the anticoagulant activity as an anti-thrombocyte activity test for the extracts of Fusarium exigua and their fractions. The antithrombotic activity of each extract was evaluated in accordance with 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 purchased from commercial plasma. The thrombin time, prothrombin time and apathy measurement were performed as follows.

Thrombin time( 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 the sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes at 37 ° C., After the addition, 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 32.1 sec. 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( 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 18.1 sec. 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 55.1 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 4] rowan Fruit extract and these Fraction Anti-thrombosis activation

As shown in Table 4, the anti-thrombocyte activity of immature and maturing of the rowers was not correlated with the coagulation inhibitory activity in the extracts regardless of the stage of maturation of the fruits, The extracts showed no blood coagulation inhibitory activity. However, in the fraction of the extract, regardless of the extraction solvent, the ethyl acetate fraction showed very strong blood coagulation inhibitory activity, and it was confirmed that the inhibition of thrombin and prothrombin associated with thrombus formation as well as the inhibition of blood coagulation factors were strong . In addition, good blood coagulation inhibitory activity was also confirmed in the butanol fraction of the hot water and ethanol extract. It is considered that the extract of Aspergillus oryzae has a weak antithrombotic activity. Ethyl acetate fraction, which is a purified product thereof, has very strong antithrombotic activity and it is considered that aspirin, which is a conventional antithrombotic agent showing side effects such as gastrointestinal disorders, It is considered that commercial antithrombotics can be developed using active fractions of fruit.

Example 3: rowan Fruit extract and sequential organic solvent Fraction Evaluation of human platelet aggregation inhibitory activity

Table 5 shows the results of evaluation of platelet aggregation inhibitory activity as a part of evaluation of anti-thrombotic activity of Fusarium oxysporum extract and fractions thereof. 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 Inhibitory Activity Platelet aggregation inhibition activity )

Platelets were purchased from the Andong National University Bio-ethics Committee (Andong National University, Daejeon, Republic of Korea) -157. The platelets were washed 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). 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 5] rowan Fruit extract and these Fraction Platelet aggregation inhibitory activity

(Sample concentration: the concentration is 0.25 mg / ml when not indicated otherwise)

As shown in Table 5, aspirin used in clinical practice as a platelet aggregation inhibitor inhibited platelet aggregation in a concentration-dependent manner. In this experiment, 50% inhibition concentration of aspirin was calculated to be 0.372 mg / ml. On the other hand, hydrothermal extracts and ethanol extracts from the matsutake matured matrices exhibited platelet aggregation ability of 124.7% and 165.4% aggregation at a concentration of 0.25 mg / ml, thereby partially promoting platelet aggregation. The hot - water extract fractions showed no significant platelet aggregation inhibitory activity, but the hexane fraction and ethyl acetate fraction of the ethanol extract showed strong inhibition of aggregation. In particular, the 50% inhibition concentration of hexane fraction was calculated as 0.168 mg / ml, and the 50% inhibition concentration of ethyl acetate fraction was calculated to be 0.218 mg / ml. As a result, 1 / 2.2 and 1 / 1.7 as compared with aspirin. These results show that the hexene and ethyl acetate fractions or the mixture thereof of the ethanol extract of the R. melanogaster can be used as an antiplatelet agent capable of replacing aspirin exhibiting adverse effects such as gastrointestinal disorders.

Example 4 Human Hematopoietic Activity of Reticular Fruits Extract and Their Fractions The human erythrocyte hemolytic activity was evaluated in order to evaluate the possibility of acute toxicity of the extract of Fruits of Raspberry, The results are shown in Table 6. 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 6] rowan Fruit extract and these Fraction Human erythrocyte hemolytic activity

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, extracts and fractions of R. melanogaster showed almost no erythrocyte hemolysis up to a concentration of 5 mg / ml and thus did not show acute toxicity and erythrocyte hemolytic activity. The above results indicate that the active fraction of Fusarium oxysporum extract can inhibit blood coagulation-related enzymes and coagulation factors without acute toxicity, exhibits excellent antithrombotic activity through inhibition of platelet aggregation, and can contribute to blood circulation improvement .

Example 5: rowan Of fruit extract of ethanol Hexen Fraction And ethyl acetate minute Chemical Characteristics and Stability of Stroke

The hexane fraction and the ethyl acetate fraction of the ethanol extract of Angelica gigas Nakai obtained in Example 1 were tested for plasma stability, thermal stability and acid stability against antithrombotic activity. The regulated active substances showed no significant inhibition of blood clotting and platelet aggregation inhibition activity even after 1 hour heat treatment at 100 ° C, 1 hour treatment at pH 2 (0.01M HCl), 1 hour treatment in plasma, Respectively. As a result of the analysis of the components of the fraction of Example 1, considering the fractionation characteristics of the organic solvent and the above stability results, the platelet aggregation inhibiting activity of the hexane fraction is expected to be a fat-soluble oil component of the rowan berries, It is expected to be a glycoside of the compound.

Claims

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Sorbus commixta A blood coagulation inhibitor containing hydrolyzate of matured or ethyl acetate fraction of ethanol extract as an active ingredient.

Platelet aggregation inhibitor containing hexane fraction of ethanol extract of Maturin ( Sorbus commixta ) matured as an active ingredient.

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