KR101471850B1 - A pharmaceutical composition comprising the hexane fraction of gardenia jasminoides extract as an effective component for anti-platelet aggregation and a health functional food comprising the same - Google Patents

Abstract

The invention gardenia (Gardenia jasminoides The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating Diseases related to thrombosis which contains the hexane fraction of an extract of Ellis as an active ingredient, The present invention relates to a pharmaceutical composition for preventing or treating a thrombotic disease which contains a fraction as an active ingredient, and a health functional food containing the hexane fraction. The pharmaceutical compositions and health functional foods for the prevention or treatment of thrombotic diseases containing the hexane fraction of the gardenia extract of the present invention as an active ingredient show strong platelet aggregation inhibiting ability as demonstrated by the examples of the present specification, In particular, the hexane fraction of the gardenia extract, which is an effective ingredient of the pharmaceutical composition and the health functional food of the present invention, shows no acute oral toxicity, has excellent thermal stability, and has a pH of 2 It is very useful 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, since no loss of platelet aggregation inhibitory ability occurs in acidic conditions and plasma. It is an invention.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a pharmaceutical composition for inhibiting platelet aggregation comprising a hexane fraction of a gardenia extract as an active ingredient and a pharmaceutical composition for preventing platelet aggregation by inhibiting platelet aggregation, SAME}

The invention gardenia (Gardenia jasminoides The present invention relates to a pharmaceutical composition and a health functional food for preventing or treating Diseases related to thrombosis which contains the hexane fraction of an extract of Ellis as an active ingredient, The present invention relates to a pharmaceutical composition for preventing or treating a thrombotic disease which contains a fraction as an active ingredient, and a health functional food containing the hexane fraction.

As a constituent of human body, blood travels through blood vessels and transports oxygen, nutrients, waste products, buffering action, maintaining body temperature, controlling osmotic pressure and ion balance, maintaining water constant, controlling liquid, And so on. Blood is in equilibrium with the coagulation and dissolution reactions, and no blood clots are formed during normal blood circulation. However, when blood clots are formed due to external blood vessel damage, internal blood clotting factors, and imbalance of thrombolytic factors, blood vessels become clogged and blood circulation is disturbed. In this case, important functions of the blood do not work and the human body is seriously damaged. Especially recently, the increase of the elderly population and the increase in the number of adult diseases, thrombosis and hypertension increases, cardiovascular and cerebrovascular diseases are increasing, so it is urgent to develop preventive and therapeutic drugs to inhibit excessive thrombogenesis.

The blood clotting reaction is caused by the adhesion of blood platelets to the blood vessel wall due to vascular damage or an imbalance of the internal blood coagulation factor / lysis factors, and the blood coagulation system is activated after the coagulation of the blood platelets to form a fibrin thrombus around the platelet aggregation mass. At this time, thrombin is activated through several steps of blood coagulation factors to produce fibrin monomers from fibrinogen, fibrin monomers are polymerized by calcium to bind to platelets and endothelial cells, cross-linked by factor XIII Forming a fibrin polymer and producing a permanent thrombus. Enzymes, factors and substances exhibiting inhibition of platelet aggregation associated with thrombus formation can be used as preventive and therapeutic agents for various thrombotic diseases caused by excessive blood coagulation. Until now, various anticoagulants such as heparin, coumarin, aspirin, and europine have been used for the prevention and treatment of thrombotic diseases. However, they are very expensive and have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity And the use thereof is limited. Recently, the development of antithrombotics from natural products derived from edible and medicinal crops having safe and antithrombotic activity has been intensively developed. Especially, ajoene of garlic, flavonoid of ginkgo leaf, rosemary essential oil, Aescin, and the like.

Meanwhile, Gardenia jasminoides Ellis) is an evergreen broad-leaved shrub of the macaques and is mature oval-shaped fruit from September to November. When it is reddish yellow, it is picked and dried to be used for medicine and dye. Especially in oriental medicine, it is used for diuretic, antipyretic, sedative and eye diseases, jaundice, etc., and is used as a hemostatic agent. The components of gardenia include genipin, geniposide, gardenoside, iridoid glycosides, crocin, crocetin, gardanin flavonoid, choline and ursolic acid.

In the past, there have been a lot of researches about the pigmentation using the gardenia (Kim Hee-gu et al., Korea Food Research and Nutrition Society, 1998, 11: 68-71) and the gardenia pigment pickle (Kim Ae Jung et al., 2008. Korean Journal of Food Science and Nutrition, , Pp. 524-529), and the addition of gardenia (Kim Mi-rim, 2006. Korean Society of Food Science and Technology, 22: 237-243) In recent years, studies on useful physiological activities of gardenia have been concentrated. In other words, the protective effect of gardenia (Chen YH et al., 2012. World J Gastroenterol. 18: 7158-7165), neuronal protective effect (Choi SJ et al., 2007. Mol Cells. 24: 113-118) (Peng CH et al., 2005. Curr Cancer Drug Targets. 5: 299-305), inhibitory effect on angiogenesis (Koo HJ et al., 2004. Planta Med. 70: 467-469) (Peng K et al., 2012. Bioorg Med Chem Lett. S0960-894X (12) 01551-X], mitigating effect of skin barrier damage (Sung Hee et al., 2010. Korean Journal of Beauty Medicine 6: 65-72), inhibition of tyrosinase enzyme activity And antioxidant activity (Pham TQ et al., 2000), and inhibition of melanogenesis (Kwak JH et al., 2004. Korean Biotechnology Journal, 19: 437-440), monoamine oxidase inhibitory activity The results showed that the iridoid genipin and the water-soluble carotenoid pigment crocin from the hot water extract of the gardenia showed antithrombotic activity Though these substances have been reported to exhibit anti-thrombogenic activity by inhibiting platelet aggregation, there has been no report on antithrombotic activity of lipid soluble in hexene from the gardenia until now, and in particular, inhibition of platelet aggregation The antithrombotic activity through activity is not known.

On the other hand, patents related to gardenia include active oxygen scavenging activity and antiviral activity of the gardenia in Korean patent No. 10-0543897 (registered on January 10, 2006) "New gardenia extracts and novel compounds isolated from gardenia" And a lipase inhibitor including a gardenia extract, a functional food and a pharmaceutical composition containing the same, a Korean Patent Registration No. 10-1078143 (registered date, "Composition for preventing and treating pancreatitis containing an extract of Gardenia japonica as an active ingredient", Korean Registered Patent No. 10-1165716 (registered date, Jul. 07, 2012) "Allergy containing a fraction of gardenia extract as an active ingredient Composition for preventing or treating disease ". Recently, Korean Patent No. 10-1089637 (registered date, November 29, 2011) entitled " Method for producing an extract of gardenia having increased antiinflammatory activity by enzyme treatment ", Korean Patent Registration No. 10-0496850 (registered date, Jun. 14) "Whitening cosmetic composition containing gardenia extract", Korean Patent No. 10-0787765 (registered date, December 13, 2007) "Composition for preventing or treating Alzheimer's disease including palmitic acid" 10-0692402 (filed on Mar. 23, 2007), "Pharmaceutical composition for prevention and treatment of depressive disorder including gardenia extract ". However, no patent has been reported on the antithrombotic agent of the gardenia extract, especially the component of the lipid-soluble hexene fraction of the gardenia.

Zhang H. Y. et al., 2013. Pharmaceutical Biology, 51: 221-225

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 a platelet aggregation inhibiting activity containing a liposoluble active ingredient extracted from a gardenia, A pharmaceutical composition for the prevention or treatment of sexual diseases, and a health functional food comprising the hexane fraction.

In order to solve the problems as described above, the present invention gardenia (Gardenia jasminoides The present invention provides a pharmaceutical composition for the prevention or treatment of a thrombotic disease comprising an effective amount of a hexane fraction of an Ellis extract.

The gardenia extract is preferably extracted with a solvent selected from the group consisting of water, alcohol, and anhydrous or hydroalcohol having 1 to 4 carbon atoms.

The gardenia extract is preferably extracted with ethanol.

The thrombotic disorder may be selected from the group consisting of acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dysesthesia, sensory abnormality, personality change, visual disturbance, epileptic seizure, pulmonary thrombosis, deep vein thrombosis, , Pain, acute peripheral arterial atresia, deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous thrombosis and central retinal vein occlusion.

The present invention also provides a health functional food comprising the hexane fraction of the gardenia extract of the present invention.

The pharmaceutical compositions and health functional foods for the prevention or treatment of thrombotic diseases containing the hexane fraction of the gardenia extract of the present invention as an active ingredient show strong platelet aggregation inhibiting ability as demonstrated by the examples of the present specification, In particular, the hexane fraction of the gardenia extract, which is an effective ingredient of the pharmaceutical composition and the health functional food of the present invention, shows no acute oral toxicity, has excellent thermal stability, and has a pH of 2 It is very useful 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, since no loss of platelet aggregation inhibitory ability occurs in acidic conditions and plasma. It is an invention.

FIG. 1 is a graph showing the results of evaluating human platelet aggregation inhibitory activity of ethanol extract of the gardenia and fractions thereof. (DMSO), 2: Aspirin (0.50 mg / ml), 3: Aspirin (0.25 mg / ml), 4: Gardenia ethanol extract (0.25 mg / ml), 5: (0.25 mg / ml), 6: ginger ethyl acetate fraction (0.25 mg / ml), 7: ginger butanol fraction (0.25 mg / ml) and 8: ginger water residue (0.25 mg / ml).

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition and a health functional food which contain a hexane fraction of a gardenia extract as an active ingredient and can exhibit a preventive or therapeutic effect of a thrombotic disease through an activity of inhibiting platelet aggregation.

The inventors of the present invention recovered a hexane fraction having the ability to inhibit platelet aggregation from a gardenia extract obtained by a certain method and confirmed that these components have excellent thermal stability and acid stability without showing oral acute toxicity, As a pharmaceutical composition and health functional food for the prevention or treatment of thrombotic diseases.

Specifically, the present inventors prepared a pharmaceutical composition and a health functional food for preventing or treating thrombotic diseases using a gardenia, preparing an ethanol extract of gardenia and extracting it with hexene to obtain a hexene fraction, Thrombin time, prothrombin time, and activated partial thromboplastin time (aPTT) and platelet aggregation inhibition were evaluated for human plasma and human thrombin, respectively. As a result, it was confirmed that platelet aggregation inhibition effect of aspartic acid (commercial antithrombotic agent), which is currently used in the hexane fraction of ethanol extract of gardenia, is stronger than that of commercially available antithrombotic agent. In addition, the hexane fraction of the gardenia extract was excellent in heat and acid stability, and acute toxicity was not observed when administered orally to rats, and the present inventors completed the present invention by confirming that the fractions were not toxic.

Accordingly, the present invention provides a pharmaceutical composition and a health functional food for the prevention or treatment of a thrombotic disease containing the hexane fraction of the gardenia extract as an active ingredient.

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

The present invention provides a method for preparing an extract, comprising: preparing an extract from a gardenia; Adjusting the hexane fraction from the gardenia extract; Checking the efficacy and stability of the extract and fraction; And examining the acute toxicity of the hexene fraction.

The "gardenia extract" contained in the composition of the present invention is obtained by extracting commercial dried gardenia with water, an organic solvent or a mixed solvent of water and organic solvent, filtering the extract using a filter net of 0.06 mm or less, ≪ / RTI > The solvent used in the present invention is water (cold water or hot water); An organic solvent such as alcohol, alcohol, or anhydrous or hydrous alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol); Or a mixed solvent of water and an organic solvent, and most preferably ethanol that can be used for the production of food ingredients.

In the present invention, first, the ginger ethanol extract and each fraction were prepared to measure thrombin time, prothrombin time, and apathy time. As a result, it was confirmed that the thrombin time at the concentration of 5 mg / ml of the gardenia ethanol extract was 1.4 times that of the no-added solution, and the prothrombin time and apathy time showed similar blood coagulation time to the no-added solution. In addition, the hexane fraction and the water residue of the ethanol extract showed no blood coagulation activity at the concentration of 5 mg / ml. However, in the case of the butanol fraction and ethyl acetate fraction, the concentration of 5 mg / ml of thrombin time and apathy Time. Especially, at 5 mg / ml concentration of ethyl acetate fraction, prothrombin time was increased to 2.4 times, prothrombin time was 1.8 times, and apathy time was 2.1 times longer than that of no - added.

Next, the platelet aggregation activity of the ethanol extract of the gardenia and the respective fractions thereof was evaluated. As a result, the hexane and ethylacetate fractions showed strong agglutination inhibition activity in the gardenia ethanol extract and the fractions thereof, and the ethanol extract, butanol fraction and water residues showed the effect of promoting flocculation. Especially, the hexane fraction showed 4 ~ 8 times inhibitory activity of aspirin at the same concentration. These results indicate that the hexane fraction of the gardenia extract has the effect of inhibiting thrombogenesis in a way that acts on human platelets and suggests that anti-thrombotic agents such as aspirin, which are reported as side effects such as gastrointestinal disorders, can be substituted .

In particular, antiplatelet activities such as genipin, geniposide and crocin isolated from the ethyl acetate fraction of ginger have recently been reported (Zhang HY et al., 2013. Pharmaceutical Biology, 51: 221-225; Thushara RM et al., 2013. Mol. Cell Biochem. : 73-83). As a result, the presence of these components in the hexane fraction was examined. However, it was confirmed that genipin, geniposide and crocin were not detected in the components of the hexane fraction of the gardenia. As a result, It is anticipated that lipid-soluble substances with anti-aggregation activity will be included and that the fractions are available as novel anti-thrombogenic compositions.

The hexane fraction of the gardenia extract of the present invention can be made into powder by a conventional powdering process such as vacuum distillation and freeze-drying, or spray drying. They maintain their activity even at 100 ° C heat treatment and pH 2 in human body.

The hexane fraction of the gardenia extract 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 can include deep vein thrombosis, portal vein thrombosis, acute renal vein thrombosis, cerebral sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition containing the hexane fraction of the gardenia extract of the present invention may be formulated into oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, sterilized injections Solutions, injections, and the like, and they can 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 water, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, 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 pharmaceutical composition 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 preferred embodiment, the effective amount of the hexane fraction of the gardenia 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 from 1 to 5,000 mg, preferably from 100 to 3,000 mg per kg of body weight 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 preventing or improving thrombotic diseases. The food containing the hexane fraction of the gardenia extract of the present invention may be in the form of a powder, a granule, a tablet, a capsule, or a beverage, for example, various foods, beverages, gums, tea, vitamin complex, Can be used.

The hexane fraction of the gardenia extract of the present invention can generally be added in an amount of 0.01 to 15% by weight of the total food, and the health beverage composition can 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 flavor agent include tau martin, rebaudioside A, glycyrrhizin, 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 gardenia extract 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: Gardenia extract and organic solvent Fraction  pharmacy

Chinese ginseng powder was purchased from Tokyo General Trading Company and pulverized and powdered. Then, 10 times volume of ethanol (95%, Duksan, Korea) was added to the germanium powder and extracted three times at room temperature for 2 days. The extract was concentrated under reduced pressure to prepare a powder, which was stored at low temperature until use. For the preparation of the fractions, the extract was suspended in distilled water and then sequentially fractionated with hexene, ethyl acetate and butanol to finally obtain a water residue.

The yields of ethanol extracts were 28 ± 2% and the yields of hexane fraction, ethyl acetate fraction, butanol fraction and water residue of the gardenia extract were 16.7%, 14.5%, 50.2% and 15.4%, respectively. Unlike conventional herbal medicine extracts, the amount of butanol fractions was very high, and a large amount of hexane fraction was obtained. Especially in the case of the hexane fraction, it means that about 4.65 g is obtained for 100 g of the gardenia, which is higher than the yield of the ethyl acetate fraction containing the conventional antithrombotic components crocin, genepin and geniposide.

The total flavonoid, total polyphenol, total sugar and reducing sugar content of the prepared gardenia extracts and fractions were measured. To determine total flavonoid content, each sample was stirred for 18 hours in methanol, 400 ml of 90% diethylene glycol was added to 400 μl of the filtered extract, 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. Total polyphenol content was determined by adding 50 μl of Folin-ciocalteau, 100 μl of Na ₂ CO ₃ The saturated solution was added and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 725 nm. Tannic acid was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was quantified by phenol-sulfuric acid method. The results are shown in Table 1 below.

Yield and Components Analysis of Ethanol Extracts and Fractions of Gardenia Extract / fraction Extraction and
Fraction efficiency (%) Content (mg / g) Total polyphenol Total flavonoid Per capita Reducing sugar Ethanol extract 28.0 ± 2.0 9.1 ± 0.15 28.5 ± 0.11 287.2 ± 5.84 108.2 ± 5.41 Hexene fraction 16.7 ± 1.1 4.6 ± 0.04 0.2 ± 0.01 9.8 ± 0.57 1.8 ± 0.28 Ethyl acetate fraction 14.5 ± 1.4 15.9 + 0.13 86.8 ± 7.18 157.0 + - 8.40 23.6 ± 1.13 Butanol fraction 50.2 ± 1.6 8.7 ± 2.15 22.9 ± 0.16 418.2 ± 11.13 109.0 + - 5.06 Water residue 15.4 ± 0.4 8.2 ± 1.41 5.9 ± 0.07 376.8 + - 8.71 277.5 ± 1.28

As shown in Table 1, the organic solvent fraction of the gardenia ethanol extracts accounted for more than 50% of the butanol fraction, the remaining fractions and the water residues of 14.5 to 16.7%, and the hexane fraction of 16.7% , And it was confirmed that the gardenia extract contained a large amount of oil components. Total polyphenol content and total flavonoid content were as follows: ethyl acetate> butanol> water residues> order of hexane fractions, total sugar content of butanol> water> ethyl acetate> order of hexane fractions, reducing sugar content water> butanol> ethyl acetate> hexene Fractions. Therefore, it can be seen that the gardenia extract contains high concentrations of polyphenols and flavonoids, and ethyl acetate and butanol fractions contain significant amount of polyphenols and flavonoids. However, the hexane fraction exhibited a very low polyphenol component and flavonoid component and thus exhibited excellent antithrombotic activity, while other useful activities such as antioxidant activity were found to be insignificant. Thus, these results indicate that the hexane fraction exhibits antithrombotic activity by previously unknown components.

Example  2: Gardenia extract and Fraction Anti-thrombosis  Activity evaluation

The antithrombotic activity of the gardenia extract and the organic solvent fraction of Example 1 was examined. Thrombin time, prothrombin time, and apathy time were measured.

Antithrombotic activity was assessed in accordance with previously reported methods (Sohn et < RTI ID = 0.0 > 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 prepared from the whole blood of the applicant, and immediately after collection, the plasma was separated by centrifugation at 5,000g for 5 minutes at 4 ° C and stored frozen (fresh frozen plasma) and thawed at room temperature if necessary. The thrombin time, prothrombin time, and apathy measurements were performed as follows.

Thrombin  time( Thrombin Time )

50 [mu] l 0.5 U thrombin (Sigma Co., USA) and 20 mM CaCl ₂ 50 μl, and 10 μl of various concentrations of sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) and allowed to react for 2 minutes. Then, 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 32.1 sec. In the case of thermal stability measurement, sample solutions at various concentrations were heat-treated at 100 ° C for 30 minutes, allowed to stand at room temperature for 1 hour, and residual activity was measured. The thrombin time measurement was expressed as the average value of the experiments repeated three or more times.

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), incubate 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 time measurement was expressed as the mean value of the experiments repeated three or more times.

aPTT  ( activated Partial Thromboplastin Time )

After adding 100 μl of plasma and 10 μl of various concentrations of the sample to a tube of Amelung coagulometer KC-1A (Japan), the mixture was heated at 37 ° C for 3 minutes, then 50 μl of aPTT reagent (Sigma, ALEXIN ^™ ) And warmed for a minute. 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

The results of measurement of blood coagulation inhibitory activity of gardenia are shown in the following [Table 2].

Antithrombotic Activity of Gardenia Ethanol Extract and Its Fraction sample Concentration (mg / ml) Thrombogenic time (seconds) Thrombin time Prothrombin time Apathy time DMSO (solvent) - 32.1 ± 0.5 18.1 ± 0.3 55.1 ± 1.4 aspirin 5.0 > 480 > 280 > 800 1.5 60.9 ± 1.8 30.7 ± 1.2 77.1 ± 2.8 Ethanol extract 5.0 44.9 ± 2.2 18.1 ± 0.5 55.1 ± 1.7 Hexene fraction 5.0 35.3 ± 1.9 18.0 ± 0.7 55.1 ± 2.4 Ethyl acetate fraction 5.0 77.1 ± 5.5 32.6 ± 3.8 115.7 ± 10.5 Butanol fraction 5.0 54.5 ± 2.3 18.1 ± 0.5 82.6 ± 5.1 Water residue 5.0 41.7 ± 1.6 18.1 ± 1.1 77.2 ± 3.5

As shown in the above Table 2, aspirin (trade name: Protect) used as a control was added at a concentration of 5 mg / ml to the thrombin time, prothrombin time, and apitime time, And 1.5 mg / ml, respectively. The blood coagulation time was 1.9 times, 1.7 times, and 1.4 times longer than that of the non-additive group, respectively. On the other hand, the ethanol extract of the gardenia showed a selective anticoagulant activity of the gardenia by selectively extending the thrombin time by 1.4 times at the concentration of 5 mg / ml. The hexane fraction of each fraction had little effect on blood coagulation. The ethyl acetate fraction of which antithrombotic activity was known was 2.4 times, 1.8 times, and 2.1 times longer than thrombin time, prothrombin time and apathy time, respectively . Butanol fraction and water residues showed weak antithrombotic activity.

Example  3: Gardenia extract, sequential organic solvent Fraction  Human platelet aggregation inhibitory activity

The anti-aggregation inhibitory activity against human platelets was evaluated in accordance with the following method as an evaluation of antithrombotic activity of the gardenia extract and fractions, and the results are shown in Table 3 and FIG.

Platelet Aggregation Inhibitory Activity Platelet aggregation inhibition activity )

Platelets were mixed with 3.2% sodium citrate solution in a ratio of 1: 9 from healthy human whole blood, and centrifuged at 1,100 rpm for 10 minutes to obtain upper layer platelet rich plasma (PRP). 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). 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 , 025% gelatin, pH 7.4), centrifuged at 3,000 rpm for 10 minutes After separation, 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.

Human Platelet Aggregation Inhibitory Activity of Gardenia Ethanol Extract and Its Fraction Sample / Control burglar
(Amplitude: ohm) inclination
(Slope) Extension time
(Lag time: seconds) Fall area
(Area under) DMSO (solvent) 20 3 22 135.9 Aspirin (0.50 mg / mL) 7 One 42 42.4 Aspirin (0.25 mg / mL) 14 2 40 80.1 Ethanol extract (0.25 mg / mL) 27 9 4 267.9 The hexane fraction (0.25 mg / mL) 4 One 47 22.2 Ethyl acetate fractions (0.25 mg / mL) 4 One 42 28.6 Butanol fraction (0.25 mg / mL) 26 4 14 200.5 The water residue (0.25 mg / mL) 26 5 8 230.6

* Smaller fall area means less platelet aggregation

As shown in Table 3, the platelet aggregation of 58.9% and the aggregation of 31.1% at the concentration of 0.5 mg / ml were higher than those of the non-additive group at the concentration of 0.25 mg / ml, And the concentration dependent coagulation inhibitory activity was confirmed.

On the other hand, the ethanol extract of the gardenia showed 197% platelet aggregation at the concentration of 0.25 mg / ml and the platelet aggregation of 147% and 169% in the butanol fraction and the water residue, respectively. Therefore, it was found that tea extract, butanol fraction and water residue act as a hemostatic agent through the promotion of platelet aggregation, suggesting the use of gardenia as a hemostatic agent in the oriental medicine.

On the other hand, the hexane fraction and the ethyl acetate fraction exhibited 16.3% and 21.0% platelet aggregation at 0.25 mg / ml concentration, respectively, as compared with the non-platelet fraction, showing stronger aggregation inhibition activity than the platelet aggregation inhibition effect exhibited at aspirin 0.5 mg / ml concentration . Previously, the genepin and crocin of the ethyl acetate fraction have been reported to exhibit platelet aggregation inhibitory activity. However, the adjusted hexene fraction from the groomer used as a hemostatic agent inhibits thrombogenesis through 4 to 8 times more potent inhibition of platelet aggregation than aspirin Is a very unusual case, and purification and identification of the active substance of the hexane fraction is necessary in the future.

Thus, through this example, it was confirmed that the hexane fraction of the gardenia extract can replace the existing aspirin with the platelet aggregation inhibitor.

Example  4: Gardenia extract Hexen Fraction  stability

The hexane fraction of the gardenia extract obtained in Example 1 was examined for thermal stability, acid stability and plasma stability. Each sample showed high stability because it did not show inhibition of blood clotting factor inhibition and platelet aggregation inhibition activity even at 2 hours treatment at 100 ° C, 2 hours treatment with pH 2 (0.01M HCl) or 2 hours treatment with plasma .

Example  5: Gardenia extract Hexen Fraction  mouse One time Oral toxicity  exam

Mouse single oral toxicity was evaluated in the hexane fraction of the gardenia extract obtained in Example 1 above. First, 4-week-old rats (Slc, ICR Mouse) were supplied from Central laboratory animals and were maintained at a temperature of 23 ± 3 ° C, a relative humidity of 50 ± 10% and a light intensity of 150 ~ Hour) for 14 days. After that, the hexane fraction obtained from Example 1 was dissolved in corn oil and then administered orally at a dose of 400, 800, 1500, or 2000 mg / kg, respectively, for 3 weeks, and the survival and the pathological abnormality were observed for one week. As a control, only corn was administered orally. As a result, the survival rate was 100% and there were no sign of pathological abnormality. Therefore, the hexane fraction isolated from gardenia, which has been used for medicinal purposes, is classified as low toxic or non - toxic, and it is judged that the use of its low concentration will not cause additional problems.

Claims (5)

A pharmaceutical composition for inhibiting platelet aggregation comprising an extract of Gardenia jasminoides Ellis as an active ingredient. The method according to claim 1,
Wherein the aqueous germanium extract is extracted with a solvent selected from the group consisting of water, alcohol, and anhydrous or hydroalcohol having 1 to 4 carbon atoms. The method according to claim 1,
Wherein the germanium extract is extracted with ethanol. delete delete

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