KR20190028004A - Pharmaceutical composition comprising the pericarp extract of litchi 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 an antithrombotic composition comprising a pericarp extract of litchi (Litchi chinensis Sonn.), a tropical fruit, as an effective ingredient and, more specifically, to a pharmaceutical composition and health functional food for prevention/treatment of thrombosis by blood coagulation inhibition which comprise ethyl acetate fractions obtained by collecting the pericarp of washed and selected litchi, extracting litchi into ethanol, and then sequentially fractioning an organic solvent as an effective ingredient. The litchi pericarp extract as an effective ingredient of the pharmaceutical composition and the health functional food for prevention or treatment of thrombosis uses an extract of the pericarp discarded after eating litchi fruit to contribute to the reuse of unused resources and environment preservation. As proven by embodiments of the present invention, the litchi pericarp extract exhibits strong antithrombotic activity by inhibition of blood coagulation factors, does not exhibit hemolytic activity for human red blood cells, has excellent thermal stability, and does not exhibit loss of blood coagulation inhibition effects even in an acidic condition of pH 2 and within blood plasma. Therefore, the litchi pericarp extract is expected to be used for prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke by blood circulation improvement. The effective ingredient can be processed in various forms such as extract, powder, tablet, and pill to be prepared in forms which can be taken at any time to be very useful in the pharmaceutical and food industries.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis and a health functional food containing a rich bark extract as an active ingredient. [0002] The present invention relates to a pharmaceutical composition for preventing or treating thrombosis,

The present invention relates to an antithrombotic composition containing, as an active ingredient, a Pericarp extract of tropical fruit rich ( Litchi chinensis Sonn.). More particularly, the present invention relates to an antithrombotic composition containing a pericarp extract of Litchi chinensis Sonn. Which comprises an ethyl acetate fraction obtained by a sequential organic solvent fraction as an active ingredient, and a health functional food.

The blood as a constituent of human body has various important functions such as oxygen and nutrients, the functions of carrying and buffering 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.

The formation of fibrin clot involves multiple steps of multiple clotting factors, activating thrombin involved in fibrin clotting, ultimately producing fibrin monomers from fibrinogen, and fibrin monomers polymerized by calcium to form platelets and endothelial cells And form a cross-linked fibrin polymer by factor XIII, producing permanent thrombosis. 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, activation of prothrombin after sequential activation of factor XII, factor XI, factor XII, factor IX and factor X is finally known to activate thrombin in the endogenous thrombosis pathway, and the specific inhibition of blood clotting factor In the extrinsic thrombosis pathway, thrombogenesis is known to be caused by the activation of factor II (prothrombin), factor V, factor VII, and factor X. To date, various blood coagulation inhibitors such as heparin, coumarin, aspirin and europaine, antiplatelet agents, thrombolytic agents and the like have been used for the prevention and treatment of thrombotic diseases. However, they are very expensive and have hemorrhagic side effects and gastrointestinal disorders And the use thereof is limited due to hypersensitivity reaction and the like.

On the other hand, as the population increases, the consumption of fruits is rapidly increasing, resulting in a large amount of fruit husks and seeds being generated as garbage. Therefore, it is urgently necessary to study the reuse of spent shells and the like (Cheok CY et al., 2016, Critical Reviews in Food Science and Nutrition, http://dx.doi.org/10.1080/ 10408398.2016.1176009). Litchi chinensis sonn. Is an evergreen arborescent tree of the perennial plant of the dicotyledonous plant. It grows up to 10 ~ 15m in height. It is also called lychee (lichee, water lychee) and Chinese cherry , Fruit is used for food. It grows well in humid acidic soil in the subtropical region, it is rooted in the root of bacteria, is native to China, and is widely cultivated in Fujian, Guangdong, Guangxi, Yunnan, Sichuan and Taiwan.

Rich fruit is a red, hard shell with a succulent white flesh, with a chocolate-colored round seed in it, and the seed and skin are discarded. It is known that the best Chinese poppies in China are effective for cosmetics (Korean confectionery association, 2004, 9: 118-119). Consumption is also increasing in Korea. In the private sector, it has been known to prevent gastrointestinal disorders, digestive disorders, constipation, increase digestive function, and rich in vitamin C and potassium to help maintain antioxidant and blood pressure. , Testicular long-stomach, stomach pain, ulcer and Jinhae drugs are known to have excellent efficacy. The actual rich flesh contains a large amount of saponin and tannin and is nutritionally superior because it contains glucose, sucrose, protein, fat, folic acid, vitamins A, B, C, citric acid, It is known. However, it is known that the rich contains allergen called profilin, and it is known that hypersensitivity, seizures, syncope, and allergic reactions may occur very rarely during overdose (Fah, J. et al., 1995. Clin. Exp. Allergy, 25 : 1018-1023).

Antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, protection against hepatotoxicity, immunostimulatory activity, hypoglycemic, anticancer, antibiotic, antiviral and hyperlipidemia , J. Agric. Food Chem. 59: 1205-1209, Ibrahim S. et al., 2015. J. Ethnopharmacology, 174: 492-513). In addition, platelet aggregation inhibitory activity and slight inhibition of blood coagulation have also been reported (Sung, YY, 2012. Mol. Med. Rep., 5: 721-724). In the case of the above paper, which reported inhibition of platelet aggregation, the region of use of the rich was dried by drying a portion of the rich flesh called a flesh in one room.

In addition, as a patent related to the rich known to date, Korean Patent No. 10-1393007 [a cosmetic composition for preventing skin aging and improving skin wrinkles containing rambutane and rich extract as an active ingredient] is disclosed in Korean Patent No. 10-0969170 (Composition for prevention or treatment of fatty liver or obesity comprising a fernaceous nuclear extract) (wherein the fleece nucleus refers to a rich seed), and Patent No. 10-1117563 (composition for antioxidant containing a foliar extract) Open No. 10-2014-0118531 and WO-2014-157865 disclose a composition for preventing, ameliorating or treating obesity, which comprises an extract of a foliar nucleus and a ginseng extract as an active ingredient.

JP-2007-0215492 discloses a method for recovering polyphenol from a rich skin and a composition thereof, JP-2010-0189444 and U.S. Patent Application No. 2004-0101508 disclose a method for recovering polyphenol from [rich bark extract Use of cosmetics for protecting skin from hair stress, etc.] are known. In addition, [HAIR TREATMENT AGENT HAVING LYCHEE EXTRACT AND TAURINE] is known in International Patent Publication WO-2009-062804, and [Scalp Cosmetics] is disclosed in JP-2009-0079018. Particularly, in relation to the rich seeds, the effect of inhibiting the glycosylation of rich seeds (Japanese Patent Laid-Open Publication No. JP-2008-0088102), the urease inhibitory effect (Japanese Patent Publication JP-2011-0006333) Patent JP-2006-0335752) is known. However, up to now, no scientific research or patent literature has been known about the antithrombotic effect of the rich bark extract and its fractions due to the strong inhibition of blood coagulation factors.

JP 2007-0215492 A JP 2010-0189444 A

 Sung, YY, 2012. Mol. Med. Rep., 5: 721-724. Antiplatelet, anticoagulant and fibrinolytic effects of Litchi chinensis Sonn. extract.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide an ethanol extract from the skin of a rich fruit that has been discarded, As an active ingredient, to provide an anti-thrombogenic composition.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which comprises a bark extract of Litchi chinensis Sonn. As an active ingredient.

The above-mentioned rich bark extract is preferably an ethyl acetate fraction of the rich-skinned ethanol extract.

The present invention also provides a blood coagulation inhibitor comprising an ethyl acetate fraction of an extract of Litchi chinensis Sonn. Shell as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving thrombosis comprising an extract of Litchi chinensis Sonn.

The above-mentioned rich bark extract is preferably an ethyl acetate fraction of the rich-skinned ethanol extract.

The pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the rich bark extract as an active ingredient of the health functional food can contribute to the reuse of insoluble resources and the preservation of the environment by using the extract of the bark to be discarded after the edible use of the rich fruit, As demonstrated by the examples in the present specification, it is possible to provide a pharmaceutical composition which exhibits strong antithrombotic activity by inhibition of blood coagulation factors, exhibits no hemolytic activity on human erythrocytes, exhibits excellent thermal stability, And the blood coagulation factor inhibitory effect is not exhibited even in plasma, it is expected that it can be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the above active ingredient can be used as an extract, powder, And can be prepared in a form that can be taken at any time. I am so the effect will be very useful inventions the pharmaceutical industry and the food industry.

Figs. 1 to 4 show the rich fruits, husks, flesh and seeds used in the experiment, respectively.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared extracts of peel and peel of rich fruits prepared by a certain method and evaluated the antithrombotic activity of the extracts in order to test the anti-thrombogenic activity of the rich extracts. The thixotropic activity was confirmed, and then the hexane fraction, ethyl acetate fraction, butanol fraction and organic solvent fraction were recovered from the rich bark extract, and the activity thereof was analyzed. As a result, the best antithrombotic activity was confirmed in the ethyl acetate fraction , And the extracts and fractions except for the hexene fraction had excellent characteristics of heat stability and acid stability, while showing no hemolytic activity against human erythrocytes. Thus, the extract was used as a pharmaceutical composition for preventing or treating / improving thrombosis And health functional foods. Respectively.

Specifically, in order to develop a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombosis using a rich known various physiological activities in the private sector, ethanol extracts of rich flesh and skin Thrombin time, prothrombin time and inhibition of blood coagulation factor (activated partial thromboplastin time: aPTT) were evaluated by subjecting these extracts to antithrombotic activity, It was confirmed that the extract of the rich bark extract had an excellent antithrombotic activity upon anticoagulant inhibition. The extracts were prepared at a concentration of 5 mg / ml at a concentration of 1.48 times the thrombin time and 1.23 times the amount of the apathy Time extension effect. At the concentration of 7mg / ml, the effect was extended by 1.92 times and 1.38 times, respectively, compared to the non - supplemented group, showing concentration - dependent antithrombotic activity.

After sequential organic solvent fractions of the rich bark extracts, the hexane fraction, the ethyl acetate fraction, the butanol fraction and the water residue after the organic solvent fraction were prepared, evaluation of their antithrombotic activity showed that they exhibited good antithrombotic activity in all the fractions In particular, the ethyl acetate fraction exhibited a prolonged thrombin time of 1.66, a prothrombin time of 1.43 times, and an apathy time extension effect of 15 times or more as compared with the no-added group at a concentration of 5 mg / ml, And suggested the possibility of clinical application as a blood coagulation inhibitor. The above ethyl acetate fraction was able to recover 0.27 g per 100 g of the rich shell. In addition, it was confirmed that other fractions other than the hexene fraction did not show hemolytic activity on human erythrocytes and did not cause acute toxicity.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which comprises an extract of Litchi chinensis Sonn. As an active ingredient.

As the active ingredient of the pharmaceutical composition, the rich bark extract is preferably the ethyl acetate fraction of the rich bark ethanol extract.

The present invention also provides a blood coagulation inhibitor comprising an ethyl acetate fraction of an extract of Litchi chinensis Sonn. Shell as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving thrombosis comprising an extract of Litchi chinensis Sonn.

As the active ingredient of the health functional food, the rich bark extract is preferably the ethyl acetate fraction of the ethanol extract of the rich skin.

Hereinafter, the method for producing the rich bark extract of the present invention and the efficacy experiments will be described in more detail.

The inventors of the present invention have found that, in order to achieve the object of the present invention, Preparing an extract from the skin; Preparing sequential organic solvent fractions of hexane, ethyl acetate and butanol from a rich bark extract exhibiting antithrombotic activity and preparing a water residue obtained therefrom; Evaluation of the antithrombotic activity of the extract and fractions and the evaluation of the stability of the active material of the ethyl acetate fraction were carried out.

The "rich bark extract" contained in the composition of the present invention comprises a step of recovering a skin from a rich fruit, a step of extracting the skin with a solvent, a step of filtering the extract using a filter net of 0.06 mm or less, ≪ / RTI >

The solvent to be used in the present invention may be selected from the group consisting of water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol), a mixed solvent of the lower alcohol and water Most preferred is hot water, or 95% ethanol extraction.

The rich bark extract of the present invention can be further fractionated with an organic solvent of hexene, ethyl acetate and butanol sequentially or separately to obtain a hexene fraction, an ethyl acetate fraction and a butanol fraction and a water residue.

In the present invention, thrombin time, prothrombin time, and apitime time were measured at a concentration of 5 mg / ml of the rich bark extract. As a result, the effect of prolonging the blood clotting time by 1.48 times, 1.00 times, and 1.23 times And at the concentration of 7 mg / ml, 1.92 times, 1.11 times, and 1.38 times, respectively, as compared with the non-added samples, indicating concentration-dependent antithrombotic activity. Considering the prolongation of thrombin time, prothrombin time, and apathy time by 1.61 times, 1.35 times, and 1.56 times, respectively, at a 1.5 mg / ml concentration of aspirin (trade name: Protect) Able to know.

As a result of evaluating the blood coagulation inhibitory activity of the extracts of the extracts rich in organic solvents and water, the concentration-dependent inhibition of thrombogenesis was observed in all the fractions. Especially, in the ethyl acetate fraction, We confirmed the inhibition of factor and suggested the possibility of clinical application as inhibitor of blood coagulation. Therefore, it has been confirmed that the ethanol extract of the rich skin and its active fraction can be developed as an antithrombotic agent capable of replacing aspirin, which is highly likely to cause side effects such as gastrointestinal disorders.

The rich bark extract of the present invention and its active fraction materials may be made into powders through conventional powdering processes such as vacuum drying and 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 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 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 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 specific method of the present invention will be described in more detail by way of examples. The following examples are only a 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 rich bark and pulp extract

After purchasing the rich fruit of Thailand, the foreign matter was removed and separated into shells, flesh and seeds (Figs. 1 to 4), which were used in the production of ethanol extracts. Ethanol extracts were prepared by adding 10 times ethanol (95%, Duksan, Korea) to each sample and once extracting at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to prepare powders. Total polyphenols, total flavonoids, total sugars and reducing sugar content were determined by analysis of the constituents of the prepared extracts. 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 and 40 μl of 1N NaOH was added. The reaction was carried out at 37 ° C. for 1 hour and then absorbance was measured at 420 nm. As a standard reagent, rutin was used. Reducing sugar was quantified by DNS method and total sugar was quantified by phenol-sulfuric acid method.

As shown in Table 1, the extraction yield of the rich fruit bark was 14.2%, which was higher than that of the pulp of 11.4%. As a result of the total polyphenol content analysis, the bark extract was 10.8 mg / g, appear. The total flavonoid content was also 1.7mg / g in shell extract and higher than 0.7mg / g in pulp. Flesh showed very low concentration of 0.7mg / g. On the other hand, the total sugar content was similar in shell and pulp, while the reducing sugar content was in the order of shell> pulp.

Example  2: Evaluation of blood coagulation inhibition activity of rich fruit extract

The blood coagulation inhibitory activity (thrombogenesis inhibitory activity) of the rich fruit extract prepared in Example 1 was evaluated and compared with the previously reported method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et Thrombin time, prothrombin time, and apathy time were measured and evaluated in the same manner as in J. Life Science, 14, 509-513, etc., 2010. J. Life Science, 20. 922-928. Plasma was measured using a commercially available control plasma (MD Pacific Technology Co., Ltd., Huayuan Industrial Area, China), and thrombin time, prothrombin time and apitime time were measured in the following manner.

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 22.4 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 ( 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 16.5 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)

Add 50 μl of aPTT reagent (Sigma, ALEXIN ^™ ) to the tubes of Amelung coagulometer KC-1A (Japan) and incubate for 3 minutes at 37 ° C. Add 100 μl of plasma and 10 μl of various concentrations of sample extract Min. 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 50.4 sec. The results of aPTT were expressed as the mean value of three repeated experiments. The activity of inhibiting blood coagulation factor was represented by aPTT divided by the aPTT of the solvent control when the sample was added.

As shown in Table 2, aspirin (trade name: Protect), which is used as an antithrombotic agent, was prolonged by 1.61 times, 1.35 times, and 1.56 times of thrombin time, prothrombin time, and apathy time at a concentration of 1.5 mg / Activity was confirmed. The peel and flesh extract of the rich fruit showed a good anticoagulant activity in a concentration dependent manner, and the peel showed better antithrombotic activity than the flesh.

On the other hand, the rich bark extract showed a DPPH anion scavenging ability of 3.5 times of the pulp extract at the concentration of 0.5 mg / ml, and a strong antioxidative activity of 4.7 times of ABTS cation scavenging ability and 5.4 times of reducing power. Oxidative stress induces structural changes in fibrinogen, and oxidized fibrinogen is closely associated with thrombogenesis and various cardiovascular diseases (Martinez M et al., 2013. Free Radic Biol. Med. 65: 411-418; Bijak M Bijak M et al., Fitoterapia 82: 811-817), this superior antioxidant activity is thought to serve as an additional mechanism of inhibiting thrombogenesis.

Example  3: sequential organic solvent of ethanol extract of the rich shell Fraction  Preparation and analysis of their components

After preparing a large amount of the ethanol extract of the present invention, the extract was fractionated with hexane, ethyl acetate, and butanol, and finally the water residue was recovered. The extraction efficiency of ethanol extract, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction are shown in Table 3.

As shown in Table 3, the majority of the ethanol extract of the rich shell was converted into the butanol fraction (84.3% of the extract), and the water residue accounted for 7.2%. On the other hand, the hexane fraction and ethyl acetate fraction accounted for 2.6% and 1.9%, respectively. These results indicate that the ethanol extract of the rich shell contains various polar substances, and that the ethyl acetate fraction from the 100 g shell can recover 0.27 g.

On the other hand, total polyphenol content was 47.9 mg / g in the ethyl acetate fraction, and the highest content was found in the fraction of ethyl acetate. The total content of flavonoids was 11.2 mg / g in the ethyl acetate fraction. On the other hand, total sugar and reducing sugar contents were 653 ~ 692mg / g and 605 ~ 618mg / g, respectively, in butanol and water residue. Therefore, various physiological activities of the rich bark extract were expected to be exhibited in the ethyl acetate fraction. Actual analysis showed strong antithrombotic activity in the ethyl acetate fraction as described in Example 4 below.

Example  4: sequential organic solvent of ethanol extract of the rich shell Fraction  Evaluation of blood coagulation inhibitory activity

The blood coagulation inhibitory activity of the rich bark extract and its fractions obtained in Example 3 was evaluated in the same manner as in Example 2, and the results are shown in Table 4.

As shown in Table 4, the aspirin showed excellent blood coagulation inhibitory activity by prolonging thrombin time, prothrombin time, and apathy time by 1.61 times, 1.35 times, and 1.56 times, respectively, at a concentration of 1.5 mg / ml. Meanwhile, the rich bark extract showed a concentration-dependent antithrombotic activity, and the thrombin time, prothrombin time, and apathy time were extended 1.92 times, 1.11 times, and 1.38 times, respectively, at the concentration of 7 mg / ml. The fractions of the rich bark extract also showed a concentration-dependent antithrombotic activity. In particular, the ethyl acetate fraction had a 1.66-fold, 1.43-fold and a 15-fold increase in thrombin time, prothrombin time, Or more. These results suggest that the ethyl acetate fraction of the extract of the rind bark may be developed as an antithrombotic agent, particularly a blood coagulation inhibitor, through the inhibition of a strong blood coagulation factor. In addition, aspirin, which is a conventional antithrombotic agent, . In addition, the fractions are expected to contribute to studies on the inhibition mechanism of specific blood coagulation factors and are expected to be used as related reagents. The fractions exhibit excellent antioxidative and carcinogenic inhibitory activities of ethyl acetate fraction, Which is expected to contribute additionally.

Example  5: Rich fruit peel extract and Fraction  Evaluation of hemolytic activity of human erythrocytes

Rich peels are not used for food, but in China we have used peels to treat smallpox and diarrhea. Therefore, it is considered that the rich bark extract and its fractions do not have any specific toxicity. To assess the potential acute toxicity of the rich bark extracts and fractions, human erythropoietic activity was evaluated and the results are shown in Table 5. The hemolytic activity was assessed in accordance with the existing report (Jung Ho Jeong, 2014, Korean J. Microbiol. Biotechnol. 42: 285-292). In brief, 100 μl of human erythrocytes washed three times with PBS was added to a 96-well microplate 100 μl of sample solution at various concentrations was added and reacted at 37 ° C for 30 minutes. Afterwards, 100 μl of the supernatant was transferred to a new microtiter plate by centrifugation (1,500 rpm) for 10 minutes and the degree of hemoglobin leaching from hemolysis was measured at 414 nm Respectively. 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.

(%) Hemolysis = [(Abs.S-Abs.C) / (Abs.T-Abs.C)] 100

Abs. S: absorbance of sample added sphere,

Abs. C: Absorbance of DMSO furniture,

Abs . T: Triton X-100 Additive  Absorbance.

First, DMSO and distilled water used as control did not have erythrocyte hemolytic activity, and Triton X-100 showed 100% hemolysis of red blood cells at a concentration of 1 mg / ml. Amphotericin B used as an anticancer drug showed 83% hemolytic activity even at a concentration of 0.07 mg / ml. On the other hand, in the case of the rich bark extract, 7.1% of red blood hemolysis was observed at a concentration of 1 mg / ml, indicating that hemolysis was not observed. The fractions did not show hemolytic activity, but 80% hemolysis activity was observed at the concentration of 1 mg / ml in the hexane fraction. The hemolytic activity of the hexane fraction was evaluated by concentration. The concentration of hemolytic activity was 47.5% at the concentration of 0.25 mg / ml and 17.1% at the concentration of 0.125 mg / ml. Therefore, it was judged that it is preferable to adjust the use concentration in the case of the hexene fraction.

Example  6: Rich bark extract and ethyl acetate Fraction  Plasma, acid and thermal stability assessment

The plasma stability, thermal stability and acid stability of the blood coagulation factor inhibitory activity of the rich bark extract and its ethyl acetate fraction obtained in Example 3 were confirmed. The extracts and ethyl acetate fractions retained excellent activity without heat treatment at 100 ° C for 1 hour, 1 hour treatment with pH 2 (0.01M HCl), and 1 hour treatment with plasma, without loss of anticoagulant activity. Thus, considering the component analysis results of Example 3, the organic solvent fraction characteristics and the stability results, the active substance of the rich bark extract is expected to be a phenolic compound, or a glycoside thereof. These results suggest that the extracts of Rich Bark and its active fractions can be practically used as antithrombotic agents, and it may be possible to supplement and replace the aspirin reported side effects such as gastrointestinal disorders.

Claims (5)

A pharmaceutical composition for preventing or treating thrombosis comprising Litchi chinensis Sonn. Bark extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the rich bark extract is an ethyl acetate fraction of a rich-skinned ethanol extract. A blood coagulation inhibitor comprising an ethyl acetate fraction of an extract of Litchi chinensis Sonn. Litchi chinensis Sonn. A health functional food for preventing or ameliorating thrombosis comprising an extract of bark as an active ingredient. 5. The health functional food according to claim 4, wherein the rich bark extract is an ethyl acetate fraction of a rich bark ethanol extract.

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