KR101801612B1 - Pharmaceutical composition comprising the extraction of kaempferia parviflora as an effective component for prevention or treatment of thrombosis and health functional food comprising the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis which contains an extract of Kaempferia parviflora as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating / Or an ethyl acetate fraction of ethyl acetate fraction and an ethyl acetate fraction of a black ginger ethanol extract as active ingredients, and to a pharmaceutical composition and a health functional food for preventing or treating / improving thrombosis through inhibition of blood coagulation. The pharmaceutical composition for the prevention or treatment / improvement of thrombosis according to the present invention and the black ginger extract as an active ingredient of the health functional food are useful for inhibiting thrombogenesis-related enzymes and blood coagulation factors and inhibiting aggregation of platelets And exhibits excellent hemostatic activity against human erythrocytes and is excellent in thermal stability and exhibits a blood coagulation factor inhibitory effect and a thrombogenic enzyme-inhibiting effect even in an acidic condition of pH 2 and plasma. It is expected that it can be used for the prevention and treatment / improvement of thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, and the active ingredient is processed into various forms such as extract, powder, Because it has excellent effects that can be prepared in a form that can be taken at any time, It is a very useful invention in the industrial and food industries.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention or treatment of a thrombotic disease and a health functional food containing a black ginger extract as an active ingredient. }

The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating / improving a thrombotic disease containing an extract of Kaempferia parviflora as an active ingredient, and more specifically, a hexane fraction of black ginger hot water extract or The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating / improving thrombotic diseases through inhibition of platelet aggregation and inhibition of blood coagulation using ethyl acetate fraction of ethyl acetate fraction and ethyl acetate fraction of black ginger ethanol extract as an effective ingredient.

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 complementarily controlling the blood coagulation system and the thrombolysis system in the body. Among them, the mechanism of the blood coagulation system is that the platelets adhere and coagulate to form blood 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, black ginger (black ginger: Kaempferia parviflora ) is a root-tinged plant of the native plant of Thailand. The underground part of black ginger has distinctive fragrance and slight spicy taste. When cut immediately after collection, it shows purple or black inside. It is known as Thai ginseng in mass cultivation and distribution in Thailand, and it is known that it helps to maintain mental and physical stability, stable blood pressure, treats dysentery and also works against abdominal pain. Southeast Asia is being used as a folk medicine along with food ingredients for the treatment of diseases such as dry mouth, gastrointestinal disorders, cold and bacterial diarrhea as well as treatment of gout, allergies and gastrointestinal disorders. In fact, black ginger has been used as a gastrointestinal disorder treatment and analgesic in Japan, and has been used as a raw material for health functional foods having an activity of improving lipid metabolism (Murata et al., 2013. Journal of Natural Medicines 67: 719-724).

 The research on the efficacy of black ginger has been concentrated mainly in Japan and Southeast Asia, and the increase in bioenergy consumption (Matsushita Mami et al., 2015. Journal of nutritional science and vitaminology 61: 79-83), improvement of sexual ability (Temkitthawon, Prapapan, 2011. Journal of Ethnopharmacology 137: 1437 ~ 1441), antioxidant activity (Kusirisin, W et al., 2009. Medicinal Chemistry 5: 139 ~ 147), anti-obesity effect (Shimada, T, 2011. Fitoterapia 82: 1272 ~ Inhibitory activity (Azuma, T. 2011. Food chemistry 125: 471-475), xanthine oxidase inhibitory activity (Nakao et al., 2011. Biol Pharm Bull 34: 1143-1146), anti- inflammatory activity (Wattanapitayakul, SK 2007. Journal of Ethnopharmacology (Supinya Tewtrakul et al., 2008. Journal of Ethnopharmacology, 116: 191 ~ 193) have been reported, and the ethanol extract Anti-ulcer activity (C. Rujjanawate et al., 2005. Journal of Ethnopharmacology, 102: 120-122) . Antibacterial activity (Sirikhansaeng P et al., 2008. Journal of Biotechnology 136: S746 ~ S747), antifungal activity and antigenic activity (Yenjai Chavi , 2004. Fitoterapia 75: .89 ~ 92), acetylcholinesterase inhibitory activity and α-glucosidase inhibitory activity (Seo Sang Hwan, 2014. Master's Thesis, Donga University, 2009. Phytotherapy research 23: 1792 ~ 1794; Azuma, T 2011. Food chemistry 125: 471-475).

The study of blood circulation in black ginger is the only report of 2013 by Kazuya M. et al. In this case, it was confirmed that the blood flow is increased by the effect of fibrinolysis which decomposes fibrin made during the production of thrombus in black ginger, And there is no report on antithrombotic activity of black ginger in terms of prevention of blood clotting and inhibition of platelet aggregation.

On the other hand, as a patent relating to black ginger, Korean Patent Registration No. 10-1551240 entitled "Composition for skin whitening comprising black ginger extract ", and Korean Patent No. 10-1406110" A composition for improving the anti-aging and elasticity of the skin and a composition for improving wrinkles ", " Novel use of flavone-based compounds ", registered patent No. 10-1525090 , A method of processing black ginger using ultrahigh pressure treatment "in Patent No. 10-1525829, and a method for processing a new compound separated from black ginger and a method for producing the same, As an antiviral agent ", and" a composition for preventing or treating muscle diseases or improving muscle function containing camphorafib boom extract or flavone compound "in Patent No. 10-1528023 The.

Also, as disclosed in Patent Publication No. 10-2011-0013685, " Functional beverage composition comprising deep sea water, fermented black ginger extract and jujube concentrate as main components ", No. 10-2015-0040618 " And JP-0121332 discloses "Kaempferia parviflora Extract Composition, Kaempferia Extract-Containing Food And Drink And Method For Improving The Taste Of Kaempferia Parviflora Extract And JP-0029499 disclose "Fermented Kaempferia Parviflora", which is known to increase functionalities, increase absorption, and process simple black ginger. JP-0107964 (EP-2851071) As for the composition for functional enhancement, "Use of Kaempferia Parviflora Extracts Or Flavone Compound For Preventing / Treating Muscle Diseases And Improving Muscle Functions" The. In addition, U.S. Patent No. US-0148504 discloses "Novel Use Of Flavone-Based Compound" as a composition for improving skin aging and elasticity and improving wrinkles.

However, there is no known patent for an active extract of black ginger which shows strong antithrombotic activity through inhibition of blood coagulation and inhibition of platelet aggregation and is not toxic to date.

 Murata, Kazuya; Deguchi, Takahiro; Fujita, Takanori; Matsuda, Hideaki. 2013. Journal of Natural Medicines v.67 no. 4, pp.719-724.

 Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems occurring in the prior art, and it is an object of the present invention to provide a pharmaceutical composition for preventing or treating a thrombotic disease comprising black ginger extract as an active ingredient, .

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating a thrombotic disease comprising an extract of black ginger ( Kaempferia parviflora ) as an active ingredient.

The black ginger extract is preferably a hot-water extract or an ethanol extract of black ginger.

Preferably, the black ginger extract is a hexane fraction, an ethyl acetate fraction or a mixture thereof of black ginger extract.

The present invention also provides a health functional food for preventing or ameliorating a thrombotic disease containing an extract of Kaempferia parviflora as an active ingredient.

The pharmaceutical composition for the prevention or treatment / improvement of the thrombotic diseases of the present invention and the black ginger extract as an active ingredient of the health functional food are useful for inhibiting thrombogenesis-related enzymes and blood coagulation factors as well as for inhibiting thrombocytopenia Exhibits a strong antithrombotic activity due to the inhibition of aggregation, exhibits no hemolytic activity on human erythrocytes, exhibits excellent thermal stability, exhibits an inhibitory effect on blood coagulation factors and thrombogenic factors It is expected that the active ingredient can be used for prevention and treatment / improvement of thrombotic diseases such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation, It has excellent effect that it can be prepared in a form that can be taken at any time Since a very useful invention, the pharmaceutical industry and the food industry.

FIG. 1 shows human platelet aggregation inhibitory activity of black ginger ethanol extract and sequential organic solvent fractions thereof: 1: solvent control (DMSO), 2: aspirin (0.125 mg / ml), 3: aspirin (0.25 mg / ml) 6: Ethyl acetate fraction of black ginger ethanol extract (0.25 mg / ml), 7: Black ginger ethanol extract (0.25 mg / ml) (0.25 mg / ml) of ethanol extract of ginger, 8: water residue (0.25 mg / ml) of ethanol extract of black ginger after organic solvent fractionation.
(DMSO), 2: aspirin (0.125 mg / ml), 3: aspirin (0.25 mg / ml), and the like. 6: Ethyl acetate fraction of black ginger hot water extract (0.25 mg / ml), 7: Black ginger hot water extract (0.25 mg / ml), 5: Black ginger hot water extract of hexane fraction Butanol fraction (0.25 mg / ml) of ginger hot water extract, 8: Water residue (0.25 mg / ml) after the organic solvent fraction of black ginger hot water extract.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared a black ginger extract by a certain method in order to test the anti-thrombotic effect of black ginger, and the extracts and the hexane fraction, ethyl acetate fraction, butanol fraction and organic solvent fraction The extract was recovered for the analysis of antithrombotic active ingredients and the extracts and fractions were confirmed to have excellent thermal stability and acid stability without showing hemolytic activity against human erythrocytes at all, A pharmaceutical composition for the prevention or treatment / improvement of diseases, and a health functional food.

In order to develop a pharmaceutical composition and a health functional food for prevention or treatment / improvement of thrombotic diseases using black ginger which is known to have excellent blood circulation improving effect in the private sector, Ethanol extracts were separately prepared, and the extracts were subjected to sequential organic solvent fractionation with hexane, ethyl acetate and butanol, respectively, and finally, water fractions were prepared 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, it was found that the hexane fraction and the ethyl acetate fraction obtained by successively fractionating the ethanol extracts in the organic solvent fraction exhibited a good blood coagulation inhibitory activity, while the hexane fraction and the ethyl acetate fraction of the hot water extract had strong intrinsic blood clots comparable to aspirin Production inhibitory activity was confirmed. Other extracts and fractions showed very weak anticoagulant activity.

The antithrombotic activity of each of the extracts and fractions was evaluated by measuring the inhibitory effect on platelet aggregation of human platelets. As a result, it was confirmed that the ethanol extract and ethyl acetate fraction thereof, the hexane fraction and the ethyl acetate fraction of the hot water extract, The extracts and active fractions showed no hemolytic activity on human erythrocytes.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of a thrombotic disease comprising an extract of black ginger as an active ingredient.

The black ginger extract is preferably a hot-water extract or an ethanol extract of black ginger.

The black ginger extract is preferably a hexane fraction, an ethyl acetate fraction or a mixture thereof obtained from black ginger extract.

In addition, the present invention provides a health functional food for preventing or ameliorating a thrombotic disease containing black ginger extract as an active ingredient.

Hereinafter, the black ginger extract of the present invention, the method for producing each active fraction, the efficacy experiment, and the like will be described in more detail.

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

The "black ginger extract" contained in the pharmaceutical composition and the health functional food of the present invention is prepared by collecting and washing the ground portion of mature black ginger, washing it, extracting it with an extraction solvent, Filtering using a filter net, concentrating it under reduced pressure, and fractionating the extract with hexane, ethyl acetate and butanol to obtain an organic solvent fraction.

The extraction solvent used in the present invention can be selected from the group consisting of 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.

The hot-water extract or the 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, the thrombin time, prothrombin time, apytime and human platelet aggregation inhibitory activity were measured at a concentration of 5 mg / ml of black ginger extract. As a result, the blood coagulation time did not increase more than that of the no- Although there was little activity, the ethanol extract showed very strong inhibition of aggregation in platelet aggregation inhibitory activity. However, the hot - water extracts showed the effect of accelerating platelet aggregation.

On the other hand, the hexane fraction and ethyl acetate fraction of the black ginger ethanol extract showed prolonged prothrombin time and apathy time in a concentration dependent manner, exhibiting strong human platelet aggregation inhibition comparable to aspirin at a concentration of 0.25 mg / ml. This suggests that the ethanol extract of black ginger and its hexane fraction and ethyl acetate fraction have very strong antithrombotic activity and suggest that anticoagulants and antiplatelet agents such as aspirin, which have high risk of side effects, can be substituted.

The hexane fraction and ethyl acetate fraction of black ginger hydrothermal extract prolonged the prothrombin time and apathy time in a concentration dependent manner. Especially in the case of apathy time, it showed an extension effect of 15 times or more at a concentration of 5 to 7 mg / ml And exhibited strong endogenous thrombogenic activity. The hexane fraction and ethylacetate fraction of the hot - water extract showed a strong inhibition of human platelet aggregation more than twice as much as aspirin at the concentration of 0.25 mg / ml, while the platelet aggregation ability was very unusual. This suggests that hexane fraction and ethyl acetate fraction of black ginger hydrothermal extract have very strong antithrombotic activity, suggesting that anticoagulants such as aspirin and antiplatelet agents, which are highly likely to have side effects, can be substituted.

The black ginger extract of the present invention and its active fractions can 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 thrombotic diseases. 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 preventing or improving thrombotic diseases.

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 essential ingredient, the above-mentioned active ingredient in a prescribed ratio, as well as a food-acceptable food supplementary additive such as natural carbohydrate and various flavoring agents as an additional ingredient.

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 black ginger extract and analysis of useful components thereof

In January 2015, black ginger grown in Thailand was purchased, washed with running water to remove foreign matter, and then used for the production of ethanol extract and hot water extract. To prepare the ethanol extract, 10 times ethanol was added to the black ginger sample, and the extract was repeated twice at room temperature. The extracts were collected by filtration and concentrated under reduced pressure to prepare ethanol extracts. In the case of the hot-water extract, 10 times of distilled water was added to the black ginger sample, and the mixture was heated at 100 ° C for 1 hour and then cooled. The mixture was cooled again and the above procedure was repeated once. The combined extracts were filtered and concentrated under reduced pressure. To prepare hot water extract. The results of each extraction efficiency and component analysis of the extract are shown in Table 1. 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.

As shown in Table 1, the extraction efficiency of black ginger was 3.8 times higher than that of ethanol extraction in hot water extraction. In the ethanol extraction of black ginger, the recovery rate was 1.92% in the first extraction, 0.58% in the second extraction, 0.42% in the third extraction and 0.30% in the fourth extraction, In the case of hot water extraction, the recovery rate in the first extraction was 6.57%, the recovery in the second extraction was 5.18%, the recovery in the third extraction was 0.44%, and the recovery in the fourth extraction was 0.30%. Therefore, it was confirmed that black ginger requires repeated extraction at least three times.

The total polyphenol contents of the extracts were 26.5 and 29.4 mg / g, respectively, and the total flavonoid contents were 17.4 and 11.3 mg / g, respectively. The ethanol extracts and hot water extracts showed higher contents appear. Total sugar content of ethanol extract and hot water extract was 193.0 and 251.6mg / g, respectively. In the case of reducing sugar, the ethanol extract contained 64% of the total sugar content and the hot - water extract had 84% of the total sugar content, indicating that it contains a very high content of reducing sugar.

Example 2: Blood coagulation inhibitory activity of black ginger extract

The ethanol coagulation inhibitory activity of the ethanol extract of Example 1 and the hot water extract was evaluated and the results are shown in Table 2. At this time, the evaluation method of blood clotting inhibition activity of black ginger 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)

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 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.

 As a result, the extracts of black ginger showed very little blood coagulation inhibitory activity at the concentration of 5 mg / ml, and the antithrombotic activity was recognized in the ethanol extracts by inhibition of prothrombin and inhibition of blood coagulation factors of hot - water extract. At this time, aspirin used as control was lengthened by 1.68 times, 1.40 times, and 1.38 times of thrombin time, prothrombin time, and apathy time, respectively, at a concentration of 1.5 mg / ml.

Example 3: Platelet Aggregation Inhibitory Activity of Black Ginger 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 3. 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. Is an important cell that secretes factors and initiates thrombogenesis by forming a primary hemostatic plug in association with collagen exposed by damage of endothelial cells. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombogenesis to be. 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.

As shown in Table 3, at 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 it was found that the aspirin was used as an antithrombotic agent in clinical practice. On the other hand, the extract of black ginger hydrothermal extract did not exhibit human platelet aggregation inhibitory activity at a concentration of 0.25 mg / ml and exhibited a weak aggregation promoting effect. However, the ethanol extract of black ginger showed more potent inhibitory activity against platelet aggregation than aspirin at the concentration of 0.25 mg / ml.

Example 4: Preparation of sequential organic solvent fractions of black ginger ethanol extract and analysis of their components

The black ginger ethanol extract prepared in Example 1 was 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 of ethanol extract, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction are shown in Table 4.

As shown in Table 4, most of the black ginger ethanol extract was converted to the ethyl acetate fraction and the butanol fraction, and the residual water after the hexane fraction and organic solvent fraction were only 2.53 and 3.64%, respectively. This result is very unusual, meaning that about 1.68 g of the ethyl acetate fraction and about 1.36 g of the butanol fraction can be recovered from 100 g of black ginger, which has been dehydrated. The total polyphenol content of the extracts was 1.6 times higher than that of the ethanol extracts of black ginger. The total flavonoid content was 1.5 times higher in the ethyl acetate fraction than in the black ginger ethanol extract. Total sugar and reducing sugar analyzes were higher in water residues and butanol fractions. Therefore, it was expected that the ginger extract would exhibit various physiological activities in ethyl acetate fraction and water residue. As a result of the 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 Black Ginger Ethanol Extract

The blood coagulation inhibitory activity of the black ginger hot-water extract and the fractions thereof obtained in Example 1 and Example 4 were evaluated in the same manner as in Example 2, and the results are shown in Table 5.

As shown in Table 5, at 1.5 mg / ml concentration of aspirin, prolonged thrombin time, prothrombin time, and apathy time were 1.68 times, 1.40 times, and 1.38 times, respectively, showing excellent blood coagulation inhibitory activity. Among the fractions of ethanol extract of black ginger, hexane fraction and ethyl acetate fraction showed prothrombin inhibition activity by prolonging prothrombin by 1.3 times at 5 mg / ml concentration, respectively, and the inhibitory activity was increased with increasing treatment concentration. In addition, the hexane fraction and the ethyl acetate fraction at the concentration of 5 mg / ml were increased by 1.2 times and 1.3 times, respectively, indicating the coagulation factor inhibition involved in the endogenous coagulation mechanism, and the inhibition activity was increased And exhibited good blood clotting inhibitory activity.

On the other hand, the butanol fraction of the ethanol extract of black ginger was found to have an anti - coagulant activity greater than that of aspirin by extending the aprotic time 1.58 times at the concentration of 5 mg / ml. These results suggest that the hexane and ethyl acetate fractions, which are the adjuvants of black ginger extract, can be developed as commercial antithrombotics, considering that black ginger is used for edible purposes. Jane aspirin, and so on. In addition, the fractions of black ginger are expected to exhibit excellent antioxidative and carcinostatic activities, thereby contributing to blood flow improvement and antithrombotic activity additionally.

Example 6 Evaluation of Human Platelet Aggregation Inhibitory Activity of Sequential Organic Solvent Fractions of Black Ginger Ethanol Extract

The human platelet aggregation inhibitory activity of the black ginger ethanol extract and its fractions obtained in Example 1 and Example 4 were evaluated in the same manner as in Example 3, and the results are shown in Table 6 and FIG.

As shown in Table 6 and FIG. 1, the ethanol extract of black ginger exhibited strong antithrombotic activity, and the fractions thereof showed stronger aggregation inhibitory activity than the ethyl acetate fraction. In addition, the hexose fraction, which is fat soluble, and the butanol fraction, which is relatively water-soluble rather than the ethyl acetate fraction, exhibited aggregation ability of 48.2% and 67.2% at 0.25 mg / ml concentration respectively, and the black ginger was composed of various components It was found that it contained an active substance which inhibited aggregation. On the other hand, water residues of 3.64% of the ethanol extract showed strong platelet aggregation promoting effect.

Example 7: Preparation of sequential organic solvent fractions of black ginger hot-water extract and analysis of their components

The black ginger hot-water extract prepared in Example 1 was prepared in large quantities, and then subjected to sequential organic solvent fractionation with hexane, ethyl acetate and butanol. Finally, the water residue was recovered. Table 7 shows the extraction efficiency of hot water extraction, the organic solvent fraction efficiency, and the component analysis results of the extract / fraction.

As shown in Table 7, the black ginger hot-water extract was mostly transferred to the butanol fraction and the water residue after fractionation, and the hexane fraction and ethyl acetate fraction were only 0.39 and 6.10%, respectively.

This result means that about 0.048 g and 0.76 g of the hexane fraction and the ethyl acetate fraction can be recovered from 100 g of black ginger, respectively, from which the foreign matter has been removed. The total polyphenol contents were in the order of water residues ≒ ethyl acetate fraction, hot water extract, butanol fraction and hexane fraction. In the case of total flavonoid contents, ethyl acetate fraction> water residue> hot water extract> butanol fraction> Hexane fractions. It was speculated that most of the active ingredients of the black ginger hot - water extract were converted into the fat - soluble ethyl acetate fraction and water - soluble water residues. On the other hand, total sugar and reducing sugar analysis showed high values in water residue and butanol fraction. Therefore, it was expected that the ginger extract would exhibit various physiological activities in ethyl acetate fraction and water residue. As a result of the analysis, the fractions showed strong antioxidative activity and nitrite scavenging ability.

Example 8: Evaluation of blood coagulation inhibition activity of sequential organic solvent fractions of black ginger hot water extract

The blood coagulation inhibitory activity of the black ginger hot-water extract and the fractions thereof obtained in Examples 1 and 7 were evaluated in the same manner as in Example 2, and the results are shown in Table 8. [

As shown in Table 8, among the fractions of the ethanol extract of black ginger, the hexene fraction and the ethyl acetate fraction showed prothrombin inhibitory activity prolonged by 1.43 to 1.49 times the prothrombin concentration at the concentration of 5 mg / ml, respectively, The inhibitory activity increased as the activity increased. The hexane fraction and the ethyl acetate fraction were increased by 1.79 times and 15 times, respectively, indicating the inhibition of blood coagulation factors involved in the endogenous coagulation mechanism at the concentration of 5 mg / ml, while the hexane fraction was 7 mg / ml The apathy time was extended more than 15 times. Therefore, the hexane fraction and the ethyl acetate fraction of the black ginger hot - water extract showed strong endogenous blood coagulation inhibitory activity.

On the other hand, the water residue of the hot - water extract of black ginger was found to be superior to aspirin by inhibiting blood coagulation factor by increasing apitime by 1.65 times at a concentration of 5 mg / ml. These results suggest that hexane and ethyl acetate fractions of black ginger hydrothermal extract can be developed as a commercial antithrombotic agent considering the fact that black ginger is used for edible purposes. It is considered that aspirin, which is a conventional antithrombotic agent showing side effects such as gastrointestinal disorders, It is possible to replace them. In addition, the fractions of black ginger are expected to exhibit excellent antioxidative and carcinostatic activities, thereby contributing to blood flow improvement and antithrombotic activity additionally.

Example 9 Evaluation of Human Platelet Aggregation Inhibitory Activity of Sequential Organic Solvent Fractions of Black Ginger Hot Water Extract

The human platelet aggregation inhibitory activity of the black ginger hot-water extract and the fractions thereof obtained in Examples 1 and 7 were evaluated in the same manner as in Example 3, and the results are shown in Table 9 and FIG.

As shown in Table 9 and FIG. 2, the hot-water extract of black ginger showed no inhibitory activity on platelet aggregation, but its hexane fraction and ethyl acetate fraction showed very strong platelet aggregation inhibitory activity. This strong activity maintained the coagulation inhibitory activity even at the concentration of 0.1 mg / ml and showed much better coagulation inhibition than aspirin. On the other hand, the butanol fraction and water residue of the hot - water extract of black ginger showed platelet aggregation promoting activity. These results suggest that the hexane fraction and the ethyl acetate fraction can be used as an antithrombotic agent that overcomes aspirin and there is little side effect such as gastrointestinal disorder such as aspirin when considering the edible history of black ginger.

Example 10: Human erythrocyte hemolytic activity of black ginger extract and its fractions

Black ginger is an edible plant that has been used for a long time as raw or extracted food, tonic and tincture and has been registered as a food ingredient and secured. To evaluate the possibility of acute toxicity of black ginger ethanol extract, hot water extract and fractions thereof, 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.

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, the hexane fraction of the hydrothermal extract at a concentration of 0.5 mg / ml exhibited a weak hemolytic activity of 14.9%, whereas all of the black ginger extract and its fractions showed no hemolysis until 0.5 mg / ml, Toxic and erythrocyte hemolytic activity. However, considering that amphoteracin B, an anticancer drug known to have haemolytic activity, exhibits hemolytic activity of 55% at a concentration of 0.00625 mg / ml and 93.7% at a concentration of 0.05 mg / ml, the hemolytic activity of the hexane fraction of the hot- .

Example 10: Evaluation of Plasma, Acid and Thermal Stability of Black Ginger Ethanol Extract, Ethyl Acetate Fraction and Hot Water Extract of Hexene and Ethyl Acetate Fractions of Ethanol Extract

Thermal stability, thermal stability and acid stability of the anti-thrombotic activity of the black ginger ethanol extracts obtained in Examples 1, 4 and 7 and the ethylacetate fractions thereof, the hexane fractions and the ethyl acetate fractions of the hot-water extracts were confirmed. These four samples did not exhibit any 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. Thus, the extracts and active fractions described above are expected to maintain antithrombotic activity during digestion and absorption processes and food production.

Claims (4)

A pharmaceutical composition for the prevention or treatment of thrombosis comprising, as an active ingredient, a hexane fraction or an ethyl acetate fraction of a black ginger ( Kaempferia parviflora ) hot-water extract. delete delete A health functional food for preventing or ameliorating thrombosis comprising hexane fraction or ethyl acetate fraction of black ginger ( Kaempferia parviflora ) hot-water extract as an active ingredient.

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