KR20110078606A - Anti-thrombus composition containing ginseng berry extracts - Google Patents

Abstract

PURPOSE: A composition containing ginseng fruit extract is provided to promote cAMP generation and to prevent platelet solidification. CONSTITUTION: A composition for suppressing platelet solidification contains ginseng fruit extract as an active ingredient. The composition is used for preventing or treating blood clots, cerebral apoplexy, cerebral hemorrhages, hypertension, and anemia. A method for preparing the ginseng fruit extract comprises: a step of isolating seeds from ginseng fruits and drying pericarp and pulp of ginseng fruits by sun light and hot air; and a step of extracting with water or ethanol.

Description

Anti-thrombus composition containing ginseng berry extracts}

The present invention relates to a composition for inhibiting platelet aggregation containing ginseng fruit extract, and more particularly, by containing ginseng fruit extract as an active ingredient, ginseng fruit extract plays an important role in platelet aggregation reaction in collagen-induced platelet aggregation reaction. It relates to a composition for inhibiting platelet aggregation by reducing the level of [Ca ^{2 +} ] _i , thereby inhibiting platelet aggregation by increasing the cAMP to inhibit the production of intracellular Ca ^{2 +} .

Vascular circulatory system diseases are now increasing as the leading cause of death. Platelet aggregation is usually an essential response for hemostasis. Abnormally hyperactivity, however, causes circulatory disorders such as thrombosis or arteriosclerosis. Therefore, it is thought that such disease can be reduced by suppressing the platelet aggregation reaction.

The mechanism of platelet aggregation by collagen is as follows. When collagen binds to a receptor called receptor tyrosine kinase (RTK) on the platelet membrane, tyrosine of the receptor is phosphorylated, thereby activating phospholipase C (PLC). PLC is a membrane protein phosphatidyl inositol-4,5-bisphosphate (PIP ₂ : Phosphatidylinositol-4,5-bisphosphate) to diacylglycerol (DG: diacylglycerol) and inositol-1,4,5-triphosphate (IP ₃ : inositol-1,4,5-trisphosphate). IP ₃ is ER: thereby mobilize the Ca ^{2 +} into the cytoplasm from (ER endoplasmic reticulum).

DG, on the other hand, activates DG-dependent protein kinase C (C-kinase) to phosphorylate intracellular proteins (40- or 47-kDa) (Berridge MJ and Irvine RF, Inositol phosphates and cell signaling, Nature , 1989, 341 : 197-205). DG also decomposes to produce arachidonic acid (20: 4), which acts as a precursor of TXA ₂ . TXA ₂ is an otacoid stimulant that further aggregates platelets. Furthermore, the increased calcium in the cells is myosin light chain kinase: to activate the (MLCK myosin light chain kinase) which Ca ^{2 +} / calmodulin (calmodulin) - then phosphorylates depend 20 kDa myosin light chain as a protein kinase. Calcium also activates the phosphorylation of DG-dependent proteins. Through this process, platelet aggregation occurs.

Platelets have cyclic nucleotides such as cAMP (Adenosine 3 ', 5'-cyclic monophosphate) and cGMP (Guanosine 3', 5'-cyclic monophosphate) as another mechanism. cAMP and cGMP are produced by adenylate cyclase and guanylate cyclase from ATP and GTP, respectively. They phosphorylate 22 kDa and 50 kDa peptides to inhibit calcium recruitment from ER and to inhibit the influx of calcium from the outside into the cell. It is also known to inhibit platelet aggregation by reducing the production of TXA ₂ .

When blood vessels are damaged, platelet aggregation, blood coagulation, and LDL oxidation occur continuously, which is called a thrombus. Such a thrombus is a cause of various diseases such as cardiovascular disease, hypertension, and cancer. Therefore, the development of drugs for preventing such blood clots is actively progressing. Anti-thrombotic agents developed to date include theopylline, the molsidomine, the verapamil, the nifedipine and the nitroglycerin. They promote the development of cAMP and cGMP that inhibit intracellular calcium recruitment. In addition, nonsteroidal drugs such as aspirin, imidazol, indomethacin, and the like have an antithrombotic effect by inhibiting the production of TXA ₂ . However, these drugs are causing a number of side effects, and thus efforts to find a solution in natural products continue.

Accordingly, the present inventors have found that the ginseng fruit extract, which is the ground portion of ginseng, inhibits platelet coagulation reaction by promoting the production of cAMP in platelet reaction induced by collagen.

Accordingly, it is an object of the present invention to provide a composition for inhibiting platelet aggregation by inhibiting platelet coagulation by promoting the production of cAMP.

In order to achieve the above object, the present invention provides a composition for inhibiting platelet aggregation containing ginseng fruit extract as an active ingredient.

Ginseng fruits according to the present invention the ginseng extract fruit extract on the platelet aggregation response induced by collagen and reduced the level of [Ca ^{2 +]} _i to play an important role in platelet agglutination.

The present invention relates to a composition for inhibiting platelet aggregation containing ginseng fruit extract as an active ingredient. The composition according to the present invention may be a composition for preventing or treating thrombus, stroke, cerebral hemorrhage, hypertension, anemia and migraine associated with platelet aggregation.

The ginseng fruit extract used in the present invention is prepared by extracting the flesh and skin of the ginseng fruit by removing the seed after drying with sunlight or hot air drying with water or ethanol.

The composition according to the present invention may be a pharmaceutical composition comprising a physiologically acceptable pharmaceutical carrier or diluent in addition to the ginseng fruit extract as an active ingredient, and is not particularly limited in the formulation, oral or injectable for oral administration is possible. It can also be used as a food additive.

The compositions of the present invention may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of dry powders which are dissolved in sterile, pyrogen-free water before use, orally formulated, subcutaneous, intravenous or It may be formulated into parenteral formulations such as intramuscular injection.

In the case of oral formulations, the compositions of the present invention may be used in known manner using pharmaceutically acceptable carriers and excipients, for example tablets, troches, sugar-containing tablets, aqueous or oily suspensions, dispersible flours or particles. , In the form of emulsions, soft or hard capsules, syrups or elixirs, which can be prepared in unit dosage form and in multi-dose containers to produce the finished product.

In oral formulations, tablets may be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate and sodium phosphate; Granulating agents such as corn, starch and alginic acid; Disintegrants; Binders such as starch, gelatin and acacia; And lubricants such as magnesium stearate, stearic acid and talc, in admixture with excipients usable for the manufacture of tablets. Tablets can be used uncoated or coated to inhibit absorption and degradation of the tablets in the gastrointestinal tract. For example, time-inhibiting substances, such as glyceryl monostearate and glyceryl distearate, can also be applied. Hard capsules are a compound of the present invention mixed with an inert solid diluent such as calcium carbonate, calcium phosphate and kaolin, and soft capsules are solvents such as polypropylene glycol, PEGs (polyethylene glycol) and ethanol that can be mixed with water. And the active ingredient in oil solvents such as peanut oil, liquid paraffin and olive oil.

The water-soluble suspending agent is a mixture of excipients and active ingredients suitable for the preparation of water-soluble suspending agents. Excipients include, for example, sodium carboxymethyl cellulose, methyl cellulose, hydroxy-propyl methyl cellulose, sodium alginate, polyvinyl-pi Suspending agents such as rollidone, gum tragacanth and gum acacia; Compounds condensing fatty acids such as polyoxyethylene stearate and alkylene oxides; Compounds in which alkylene oxides are condensed with long fatty acids such as heptadecaethyleneoxycetanol; Compounds which condensate partial esters derived from anhydrous hexitol anhydride and fatty acids, such as polyoxyethylene sorbitol monooleate, with ethylene oxide; Wetting agents; Or dispersing agents. The water-soluble suspending agent may further contain preservatives, colorants, spices and sweeteners.

The oily suspension is a suspension of an active ingredient in a vegetable oil such as olive oil, sesami oil or a mineral oil such as liquid paraffin. For example, a thickening agent such as beeswax, hardened paraffin and cetyl alcohol may be used. It may contain. It may also contain preservatives, colorants, spices, sweeteners, and the like, which can be preserved by adding antioxidants such as vitamin C.

Dispersible powders and particles have an active ingredient in a state of mixing together a dispersing agent, wetting agent, suspending agent and preservative. Suitable dispersing, wetting or suspending agents can be mentioned by way of example already mentioned above. Additional excipients include, for example, sweeteners, spices and colorants.

Water-in-oil emulsions are derived from vegetable oils such as olive oil or mineral oils such as liquid paraffin, and are derived from natural phospholipids such as soy bean lecithin, and esters of anhydrous hecitol or fatty acids such as sorbitan monooleate. And compounds obtained by condensation of ethylene oxide with partial esters derived from anhydrous hexitol anhydride and fatty acids, such as lyoxyethylene sorbitol monooleate, as emulsifiers.

Syrups and elixirs can be prepared by mixing the active ingredients with sweeteners such as glycerol, propylene glycol, sorbitol and sucrose.

Parenteral formulations may be injected as sterile injectable solutions or as suspensions in which the active ingredient is suspended in a non-toxic usable diluent or solvent such as 1,3-butanediol. Among the excipients or solvents that can be used are water, Ringer's solution and isotonic saline solution. Cosolvents such as ethanol, polyethylene glycol, and polypropylene glycol can also be used. In addition, sterile, nonvolatile oils can be customarily used as solvents or suspending solvents. Mixed, non-stimulating, bland fixed oils for this purpose include synthetic mono- and diglycerides. In addition, fatty acids such as oleic acid can be used to prepare the injections. The suppository form is formulated by mixing the drug with a suitable non-irritating excipient, e.g. cocoa butter, polyethylene glycol, which is solid at room temperature but liquid at the rectal temperature and will melt in the rectum to release the drug. May be administered.

Food additives may include beverages, baking, drinks, fats, ice cream, sweets, rice cakes, snacks, baby food, alcoholic beverages, seasonings, retort or canned food.

The cholesterol lowering composition of the present invention may be formulated in an oral dosage form such as a dry powder which is in the form of a solution, a suspension or an emulsion in an oil or an aqueous medium, or dissolved in sterile, water-free water before use. It may also be formulated into parenteral formulations such as subcutaneous, intravenous or intramuscular injection.

In the case of oral dosage forms, for example, tablets, troches, sugar-containing tablets, aqueous or oily suspensions, dispersible powders, etc., in a known manner using a pharmaceutically acceptable carrier or forming agent. Or in the form of oral dosage forms in the form of particles, emulsions, soft or hard capsules, syrups, elixirs, which may be suitably prepared according to unit dosages, forms and the like.

Parenteral formulations may be injected as a sterile injectable solution or as a suspension in which the active ingredient is suspended in a solvent such as a non-toxic usable diluent or 1,3-butanediol. Excipients or solvents that may be used include water, Ringer's solution, and isotonic saline solution. Cosolvents such as ethanol, polyethylene glycol, and polypropylene glycol may be used. In addition, sterile, nonvolatile oils can be customarily used as solvents or suspending solvents. The suppository form is formulated by mixing the drug with a suitable non-irritating excipient, such as cocoa butter or polyethylene glycol, which is solid at room temperature but liquid at the rectal temperature and will melt in the rectum to release the drug and then enter the rectum. Administration.

When treating a disease using the composition of the present invention, the dose of the active ingredient ginseng fruit extract is the age, weight, general state of health, sex, meal, time of administration, rate of excretion, combination of drugs, disease during treatment Depending on the degree of, etc., depending on the disease can be used every day up to 0.01 to 140 mg / kg, can be used up to 0.5 mg to 7 g per day per patient.

On the other hand, the amount of the compound of the present invention mixed with the carrier material to determine one formulation depends on the mode of administration and the patient being treated. For example, formulations intended for topical application and oral administration of skin to humans will contain from 5 to 95% by weight of carrier materials and from 0.5 mg to 5 g of active ingredient and parenteral administration to humans. Formulations intended for this purpose will contain from 5 to 99% of the carrier composition and from 0.1 mg to 2.5 g of active ingredient.

Hereinafter, the content of the present invention will be described in more detail through examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Example 1 Preparation of Ginseng Fruit Extract

1) Ginseng fruit pretreatment: Raw ginseng fruit was harvested, seed was separated and removed, and then the ginseng fruit dried raw material was prepared by sun drying or hot air drying.

2) Preparation of ginseng fruit extract: 3 g of water was added to 1 kg of dried ginseng fruit extract to extract reflux, and then filtered and concentrated under reduced pressure at 40 to 45 ° C. to obtain 300 g of ginseng fruit extract.

Comparative Example 1 Ginseng Root Extract

3 g of water was added to each kg of ginseng root to extract reflux, followed by filtration and concentrated under reduced pressure at 40 ~ 45 ℃ to prepare a ginseng root extract.

Test Example 1 Comparison of Ingredients of Ginseng Fruit Extract

<Ginsenoside (Ginseng Saponin) Analysis of Ginseng Fruit and Ginseng Root>

In Example 1 and Comparative Example 1, respectively, ginseng fruit and ginseng root extract were prepared, and then, the extracts were treated with ether to remove fat-soluble components, followed by extracting and concentrating ginsponin with butanol (BuOH). Cenoside component analysis was performed, and the results are shown in Table 1 below.

The ginseng fruit extract prepared in Example 1 had about twice the content of the ginseng root extract prepared in Comparative Example 1 in the content of irradiated phonynin, and ginsenosides were PD (Protopanaxadiol)-"ginsenoside Rb1, Rb2. And Rc and Rd "and PT (Protopanaxatriol)-" Ginsenoside Re, Rg1 and Rg2 "ratios were 0.73 and 3.23, respectively. .

Mineral Analysis of Ginseng Fruit Extract

In order to distinguish that the ginseng fruit extract prepared in Example 1 has the characteristics of 'fruit' different from ginseng, mineral components including vitamins were analyzed, and the results are shown in Table 2 below.

As described above, the ginseng fruit used in the present invention contains more ginseng saponin than the ginseng root, and at the same time, the composition of the saponin is opposite to that of the ginseng root. Could confirm.

[Test Example 2] Determination of Acetylcholine Relaxation by Example Concentration According to the Present Invention

Four-month-old hypertensive model rats (spontaneously hypertensive rats, hereinafter referred to as 'SHR') were used, and the ginseng of Example 1 according to the present invention was used as a control group of the group administered with continuous drinking water for 6 weeks to the hypertension model rats. Endothelial cell-dependent vasodilation ability compared to the group administered continuously by dissolving 100 mg / kg / day of fruit extract in drinking water for 6 weeks and the group administered with 60 mg / kg / day dissolved in drinking water for 6 weeks. Was analyzed.

Each SHR abdomen was cut under ether anesthesia, the thoracic aorta was removed, cut into 0.5 cm rings, and 37 ° C. KH (Krebs-Henseleit, pH 7.4) saturated with 95% + 0.5% -CO + O ₂ gas. Suspension was carried out in a 20 ml volume of tissue bath filled with physiological solution.

The other end of the vessel was connected to a force transducer and the contraction and relaxation responses were recorded on a physiograph. A rest tension of 2 g was given and allowed to rest for 60 minutes. Thereafter, about 10 g of norepinephrine (norephinehprione) was added to shrink about 1 g, and the relaxation reaction by acetylcholine was measured cumulatively and the results are shown in FIG. 1. Acetylcholine is an endothelial cell-dependent relaxation agent, and it has been reported that the relaxation response in the blood vessels of hypertensive SHR is higher than that of normal rat thoracic aorta.

As can be seen from the results of Figure 1, the relaxation response of acetylcholine was increased sensitively in proportion to the dose in the group administered the ginseng fruit extract of Example 1.

Test Example 3 Endothelial Cell-dependent Vasodilation Capacity

10 g of ginseng fruit extract and ginseng extract 10-5 g / ml of the control group according to the present invention and 10-5 g / ml of the extract was administered to the tissue tank, respectively, 10, 20, 30, 40, 50, 60 mM concentration of potassium chloride in each tissue tank In addition, 10 M concentration in angiotensin II 10-M was cumulatively measured and compared with each other, and the results are shown in FIGS. 2A and 2B.

As can be seen from the results of Figures 2a and 2b, the ginseng fruit extract administration group according to the present invention did not affect the dose-dependent contraction response by potassium chloride and angiotensin II.

Test Example 4 Effect of Ginseng Fruit Extract on Platelet Aggregation Induced by Collagen

<Experimental material>

Collagen used in the test example of the present invention was purchased from Chrono-Log Corporation (Harbourtown, Pennsylvania, USA). 8-Bromoadenosine-3 ′, 5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cAMPS), 9- (tetrahydro- 2′-puryl) adenine (SQ22536), 1-ethyl-4-[(1-methylethylidene) hydrazino] 1H-pyrazolo [3,4-b] pyridine-5-carboxylic acid (Etazolate ) And other reagents were purchased from Sigma Chemical Corporation, St. Louis, USA. cAMP, cGMP, TXB ₂ enzyme immunoassay kit (enzymeimmunoassay kit) used a product of Amersham Bioscience (Buckinghamshire, UK).

<Preparation of Washed Rat Platelets>

Blood was collected using 0.38% sodium citrate as an anticoagulant in the abdominal vein of normal SD rats. The collected blood was centrifuged at 220 × g for 10 minutes to obtain an upper layer platelet-rich plasma (PRP). PRP was centrifuged at 1650 × g for 10 minutes to obtain lower platelet pellets. The pellet was washed twice with wash 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 6.9). The washed platelets were suspended using suspension 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). Cell suspensions were prepared. The platelet count was then adjusted to 5 × 10 ⁸ platelets / ml. All procedures were conducted at room temperature, to rule out the effects on platelets at low temperatures (Reuter H, et. al ., Studies of the aggregation of human blood platelets. 3.On the inhibition of platelet aggregation in EDTA plasma following incubation at 37 degrees C, Klin Wochenschr , 1967, 45: 1147-1149).

<Platelet aggregation reaction by collagen concentration>

To determine the platelet aggregation reaction according to collagen concentration, CaCl ₂ in 10 ⁸ / ml After addition of 2 mM and preincubation at 37 ° C. for 3 minutes, the collagen was stimulated with 0.5, 1, 3, 5 and 10 μg / ml to measure the aggregation rate for 5 minutes, and the results are shown in FIG. 3. Platelet aggregation rate (%) was obtained by measuring the light transmittance at 660 nm of the Lumiaggregometer (Chrono-Logsa, Harbortown, Pa.), And calculates the platelet aggregation rate (%) from the light transmittance. The law is as follows.

The transmittance of light is up to 100%, and the platelet aggregation rate (%) increases as the transmittance increases. As shown in Fig. 3, the recording paper of the coagulation system is composed of 0% to 100% scale. When light penetrates the platelet-containing suspended platelet suspension to 10% of the recording paper, it is fixed to zero as a base line, and the light penetrates the platelet-free washing buffer. Set the case to 90% and fix it at the maximum transmittance. When collagen is added to the suspension of washed platelets to cause platelet aggregation reaction, from 10% point of the recording paper (baseline) to 90% point of the recording paper (maximum transmittance) As the transmittance increases, agglomeration reaction curves are drawn on the recording paper. That is, the increase in permeability from 10% of the washed platelets means an increase in the aggregation reaction rate.

However, since the permeability increased by collagen is measured in a fixed state by converting 100% permeability to 80% (90%-10% = 80%) in the recording paper of the aggregation system, the actual platelet aggregation rate (%) is expressed by the following equation. Is calculated as 1.

Maximum Platelet Aggregation (%) = A / 80 x 100

Here, A is the transmittance 5 minutes after the addition of collagen to the washed platelet suspension prepared from the blood of the rat which was administered or not drug.

In the results of FIG. 3, since the aggregation was the highest when 10 μg / ml collagen was added, all experiments used 10 μg / ml collagen.

<Effect of Ginseng Fruit Extract on Platelet Aggregation Induced by Collagen>

The measurement of platelet aggregation was performed by Born's method (Born, GVR, Aggregation of blood platelets by adenosine diphosphate and its reversal, Nature (London), 1962, 194: 927-929).

CaCl ₂ to washed platelets 10 ⁸ / ml After 2 minutes of preincubation at 37 ° C. for 3 minutes, 10 μg / ml of collagen and 0, 1, 5, 10, 30, and 50 μM of collagen were added, respectively, for 5 minutes. After proceeding to measure the aggregation rate, the results are shown in Figure 4a. At this time, a suspension buffer was used as a blank (permeability 0), and the ginseng fruit extract was used in a distilled water, so there was no effect on the solvent.

In the results of Figure 4a, it was confirmed that the platelet aggregation rate decreased in proportion to the concentration of the ginseng fruit extract when the ginseng fruit extract of Example 1 was pretreated.

Based on these results, the inhibition rate for inhibiting platelet aggregation according to the ginseng fruit extract concentration was calculated according to Equation 2 below, and the results are shown in FIG. 4B.

억제율(%) = [] × 100

% Inhibition = [] × 100

C: Coagulation rate in platelets treated with collagen only

T: Coagulation Rate in Platelets Treated with Collagen and Ginseng Fruit Extract

In the results of Figure 4b, when the ginseng fruit extract content of Example 1 is 50 μM, it was confirmed that platelet aggregation is suppressed as much as 80% compared to the test group without the ginseng fruit extract.

Test Example 5 Effect of Ginseng Fruit Extract on Intracellular Calcium Increase by Collagen

Intracellular calcium is critical for many cellular responses, including platelet aggregation. Intracellular calcium was measured using the fluorescent material fura 2-AM.

Cytoplasm in ^{Ca 2 + ([Ca 2 +} ] i) In order to measure the concentration of, by loading the fluorescent indicator dye Fura 2-AM in (fluorescent indicator dye) in the platelet fluorescence indicating the fura 2 in combination with Ca ^{2 +} (fluorescence ) Was measured using an SPF 25 fluorimeter (Kontron). The drive was performed for 8 minutes at 37 ° C with excitation of 340 nm and emission of 510 nm. At 3 minutes, 10 μg / ml of collagen was added to stimulate platelets. Actual [Ca ^{2 +]} _i is the relative fluorescence.: Grynkiewicz method of it were a (rf relative fluorescence) (Grynkiewcz G , Poenie M and Tsien RY A new generation of [Ca 2 +] indicators with greatly improved fluorescence properties, J Biol Chem , 1985, 260: 3440-3450) was used to calculate the concentration of Ca ^{2 +} ([Ca ^{2 +} ] _i ) according to the following Equation 3, and then the inhibition rate was calculated according to the following Equation 4, the results Table 3 shows.

[Ca^{2 +}]_i =

[Ca ^{2 +} ] _i =

Where K _d is the resolution constant of fura 2-AM (224 nM, 37 ° C.), F is the actual measured rf value of each sample, and F _max is the maximum fluorescence of 0.1% in the presence of 2 mM CaCl _2. Measured by addition of Triton X-100, 0.1 _min Triton X-100 and 20 mM Tris-3 mM EGTA without CaCl ₂ as F _min minimum fluorescence [mean ± SD (n = 7)].

억제율(%) = [] × 100

% Inhibition = [] × 100

C: [Ca ^{2 +]} _i in a treatment of collagen, only platelets

T: [Ca ^{2 +} ] _i in platelets treated with collagen and ginseng fruit extract

Effect of Ginseng Fruit Extract on [Ca ^{2 +} ] _i Increased by Collagen Test substance [Ca ^{2 +} ] _i (nM) % Inhibition Intact cells 48.6 ± 9.2 - Collagen (10 μg / ml) 90.2 ± 8.3 0 Collagen (10 μg / ml) + Ginseng Fruit Extract (1 μM) 77.6 ± 5.4 13.9 Collagen (10 μg / ml) + Ginseng Fruit Extract (5 μM) 70.4 ± 9.1 21.9 Collagen (10 μg / ml) + Ginseng Fruit Extract (10 μM) 66.1 ± 8.7 26.7 Collagen (10 μg / ml) + Ginseng Fruit Extract (30 μM) 55.8 ± 4.9 38.1 Collagen (10 μg / ml) + Ginseng Fruit Extract (50 μM 52.7 ± 2.6 41.6

In the results of Table 3, due to collagen stimulation, the amount of calcium in the platelets rapidly increased. However, as a result of pre-treatment with ginseng fruit extract of Example 1, in proportion to the concentration of the ginseng extract of the fruit is the concentration of [Ca ^{2 +]} _i was inhibited up to 41.6%. In other words, the concentration of [Ca ^{2 +} ] _i decreased in proportion to the concentration of ginseng fruit extract.

Test Example 6 Effect of Ginseng Fruit Extract on Collagen-induced Platelet Aggregation in the Presence of Adenylate Cycase Inhibitor or A-Kinase Inhibitor

To determine whether cAMP increased by ginseng fruit extract directly affects platelet aggregation, CaCl ₂ in washed platelets 10 ⁸ / ml in the same manner as in Test Example 1 After preincubation at 37 ° C. for 2 minutes with 2 mM addition, 10 μg / ml of collagen, 50 μM of ginseng fruit extract, 10 μM of SQ 22536, and 20 μM of Rp-8-Br-cAMPS, respectively, were allowed to aggregate for 5 minutes. After the reaction was performed, platelet aggregation rate was measured, and the results are shown in FIGS. 5 and 6.

In the results of Figure 5, the platelet aggregation reaction was significantly reduced in the group treated only ginseng fruit extract, the platelet aggregation rate increased again when the SQ22536 inhibitor of adenylate cyclase was treated. Through the results, it was confirmed that the ginseng fruit extract according to the present invention inhibits platelet aggregation by increasing the intracellular cAMP.

In addition, intracellular cAMP activates A-kinase to induce several cellular responses. Therefore, in order to investigate the effect of cAMP on the downstream A-kinase, we measured the platelet aggregation reaction when R-P-8-Br-cAMPS, an A-kinase inhibitor, was treated with ginseng fruit extract ( 6), the platelet aggregation reaction that was inhibited when only the ginseng fruit extract was treated was confirmed that the aggregation rate increased in the same manner as the SQ22536 treatment when Rp-8-Br-cAMPS treatment. In other words, by treating Rp-8-Br-cAMPS, an inhibitor of A-kinase, it was found that the platelet aggregation reaction inhibited by the ginseng fruit extract was increased again.

Test Example 7 Toxicity Test

<Oral administration>

ICR-based mice and Sprague Dawley (cod hyochang science) were divided into four groups of 10 mice each, orally administered with the extracts of Example 1 of the present invention at doses of 500, 725, 1000 and 5000 mg / kg, respectively. After two weeks of toxicity, none of the four groups died and no symptoms were found.

<Abdominal administration>

ICR mice (25 ± 5 g) and Sprague Dawley (cod hyochang science) were divided into four groups of 10 animals each, and the extracts of Example 1 of the present invention were 25, 250, 500 and 725 mg, respectively. Toxicity was observed for 24 hours after intraperitoneal administration at / kg. There were no deaths in all four groups and no symptoms were apparent in the control group. From the above results, it was confirmed that the extract of the present invention has little acute toxicity.

Hereinafter, an example of the preparation of the pharmaceutical composition will be described, but it is not intended to limit the present invention but merely to explain in detail.

Formulation Example 1 Powder

A powder containing 50 mg of ginseng fruit extract of Example 1, 100 mg of lactose and 10 mg of talc was prepared per package according to the preparation method of powder in the pharmacopeia formulation.

Formulation Example 2 Tablet

A tablet containing 50 mg of ginseng fruit extract of Example 1, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate was prepared per tablet according to the preparation method of the tablet in the pharmacopeia formulation.

Formulation Example 3 Capsule

A capsule containing 50 mg of ginseng fruit extract of Example 1, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate was prepared per capsule according to the preparation method of the capsule in the pharmacopeia formulation.

Formulation Example 4 Injection

Injectable drugs were prepared by appropriately mixing sterile distilled water and a pH adjusting agent for injection into 50 mg of ginseng fruit extract of Example 1 per ampule (2 ml) according to the preparation method of injectable drugs.

Formulation Example 5 Liquid

According to the preparation method of the liquid formulation in the pharmacopeia formulation, 50 mg of ginseng fruit extract of Example 1, 10 g of isomerized sugar, 5 g of mannitol, and purified water were mixed per 100 ml of the formulation.

Formulation Example 6 Healthy Functional Drink

According to the conventional method for preparing a health functional beverage, 1000 mg of ginseng fruit extract of Example 1, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate, 1 g of taurine, and purified water were added and mixed to make a total of 900 ml, then about 1 After stirring and heating at 85 ° C. for a period of time, the resulting solution was collected by filtration into a sterilized 2 ° C. container, sealed and sterilized and then stored in the refrigerator.

Although the composition ratio is mixed with a relatively suitable component in a preferred embodiment in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

1 is a graph showing the vasorelaxation response by acetylcholine.

2a and 2b are graphs showing vasoconstriction by potassium chloride and angiotensin II.

3 is a recording sheet of a lumiaggregometer for measuring platelet aggregation rate.

Figure 4a is a graph showing the change of aggregation rate according to the concentration of ginseng fruit extract in platelet aggregation reaction induced collagen, Figure 4b is a graph showing the inhibition of platelet aggregation according to the concentration of ginseng fruit extract [mean ± SD (n = 3 -4). ^* P <0.05, ^** P <0.01, compared with collagen induced platelets without ginseng fruit extract].

5 is a graph showing the effect of ginseng fruit extract on collagen-induced platelet aggregation in the presence of SQ22536 [mean ± SD (n = 3). ^** P <0.01, compared with collagen induced platelets added only ginseng fruit extract without SQ22536].

6 is a graph showing the effect of ginseng fruit extract on collagen-induced platelet aggregation in the presence of Rp-8-Br-cAMPS [mean ± SD (n = 3). ^** P <0.01, compared with collagen-induced platelets added only ginseng fruit extract without Rp-8-Br-cAMPS].

Claims (2)

Platelet aggregation inhibiting composition containing ginseng fruit extract as an active ingredient. The method of claim 1, The composition is a composition for inhibiting platelet aggregation, which is a composition for preventing or treating blood clots, stroke, cerebral hemorrhage, high blood pressure, anemia and migraine headaches.

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