KR20220026082A - Pharmaceutical composition comprising the root extract of cimicifuga simplex 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 comprising an extract of Cimicifuga simplex under ground part as an active ingredient for preventing or treating thrombosis and, more specifically, to a pharmaceutical composition and a health functional food comprising an extract of Cimicifuga simplex under ground part as an active ingredient for preventing or treating/ameliorating thrombosis through inhibited blood coagulation and platelet agglutination. According to the present invention, the extract of Cirsium platelet agglutination, which is an effective component of the pharmaceutical composition and health functional food for preventing or treating thrombosis, shows a strong antithrombotic activity caused by an inhibitory effect of platelet agglutination which serves to initiate thrombogenesis while inhibiting thrombogenesis-related enzymes and blood coagulation factors, and the complex fermented solution also does not show any hemolytic activity to human red blood cells has excellent thermal stability, does not show any loss of an inhibitory effect of blood coagulation factors and an inhibitory effect of thrombogenesis-related enzymes under an acid condition of pH 2 and even in plasma, and thus it is expected that the complex fermented solution will be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke through improved blood circulation, and the effective component has an excellent effect in that the component may be processed into various forms of extract, powder, pill, tablet, etc. to be compounded in a readily edible form, and thus is an invention very useful in pharmaceutical industry and food industry.

Description

Pharmaceutical composition and health functional food for prevention or treatment of thrombosis containing extract of Chotdae Equestrian underground part as an active ingredient }

The present invention relates to a pharmaceutical composition for the prevention or treatment of thrombosis and a health functional food containing the underground extract of Cimicifuga simplex as an active ingredient. It relates to a pharmaceutical composition and a health functional food for preventing or treating/improving thrombosis through inhibition of blood coagulation and platelet aggregation.

As a component of the human body, blood has a variety of important functions, such as transporting oxygen, nutrients, and wastes, buffering, maintaining body temperature, osmotic pressure and ion balance, maintaining water schedule, regulating fluidity, maintaining and controlling blood pressure, and defending the body. there is. Normal blood circulation facilitates blood circulation as the blood coagulation and thrombolytic systems in the body are controlled complementary to each other. , it has been reported that the blood coagulation system is activated to form a fibrin clot centered on platelet agglomerates.

On the other hand, the generation of fibrin thrombi is activated by thrombin involved in fibrin coagulation through a multi-step reaction of numerous blood coagulation factors, and finally produces fibrin monomers from fibrinogen, which are polymerized by calcium, so that platelets and endothelium It binds to cells and forms a fibrin polymer cross-linked by factor XIII, forming a permanent thrombus. In addition, thrombin plays a pivotal role in thrombus formation by activating platelets, factor V, and factor VII to promote a blood clotting reaction. Therefore, the thrombin activity inhibitor can be used as a very useful prophylactic and therapeutic agent for various thrombotic diseases caused by excessive blood clotting. On the other hand, it is known that the activation of prothrombin following sequential activation of factor XII, factor XI, factor IX, and factor X in the endogenous thrombus formation pathway ultimately activates thrombin. being targeted To date, various anticoagulants such as heparin, coumarin, aspirin, and urokinase have been used for the prevention and treatment of thrombotic diseases. As such, its use is limited.

On the other hand, horseback riding (升麻, black cohosh) is a perennial plant of the buttercup family, and it is similar to hemp, but it is called horseback riding because its properties increase. There are stork riding, candlestick riding, king riding, big horse riding, herb riding, and mountain candlestick riding. In Korea, three species of horseback riding ( Cimicifuga heracleifolia var. bifida ), candlestick horseback riding ( Cimicifuga simplex ), and eye horse riding ( Cimicifuga dahurica ) are widely growing wild in Korea.

Cimicifuga simplex ( Cimicifuga simplex ) is called candlestick riding because the shape of the unbranched inflorescence resembles the shape of a candlestick. It is distributed in Japan, Kamchatka, Sakhalin, and northeastern Asia, including Korea, and grows deep in forests in mountainous areas. The stem reaches a height of 1m and has distinctive white hairs along with the flower ears. Flowers bloom in June-July and are white, with male and female flowers running in raceme at the end of the stem. There is a peduncle, and fine hairs are scattered on the flower stalk. Chotdae Equestrian horses are cultivated for ornamental purposes because of their beautiful flowers, and their roots are medicinal, and young shoots are edible as greens. In oriental medicine, it is used for measles, sore throat, inflammation of the lips and gingivitis. It is known to be effective in the treatment of diarrhea and dysentery due to its antibacterial action. In Japan, the root is used as an antipyretic and detoxifying agent.

As a study on candlestick riding, antioxidant activity and nitric oxide production inhibitory activity of the underground part of candlestick riding (Cho Hyangsun, 2007. Chung-Ang University master's thesis), isolation of cycloartenol triterpenoid saponins (Wu L et al., 2015, Chin. J. Nat. Med) 13: 81-89; Kuang H et al., 2011, Molecules 16: 4348-4357; Kuang H et al., 2012. Planta Med. 78: 622-625), immune enhancing activity by cycloartenol triterpenoid saponins (Su Y et al. 2017, Bioorg. Med. Chem. 25: 4917-4923), analysis of phenolic components in above-ground parts (Iwanaga A et al., 2010, J. Nat. Prod. 73: 609-612), extraction and analysis of essential oils in underground parts (Miyazawa M, Kawata J) 2006. Nat. Prod. Res. 20: 542-547) and nucleoside transport inhibition by cimicifugoside isolated from the underground and cytotoxic enhancing activity of methotrexate used as an anticancer agent (Yawata A et al., 2009. Eur. J. Pharm. Sci. 38: 355-361) and the like are known. However, to date, there is no known report on the strong antithrombotic activity of the underground extract of Chotdae horseback riding.

In addition, as a patent related to candlestick riding, Korean Patent Registration No. 10-1303693 discloses [composition for improving fat metabolism disease, menopause disease or cardiovascular disease], which is a three-leaf riding ( Cimicifuga heracleifolia ) extract, eye riding ( Cimicifuga ). davurica ) extract, daffodil ( Cimicifuga japonia ) extract and candlestick horse riding ( Cimicifuga simplex ) Food composition patent for improving fat metabolism disease, menopause disease or cardiovascular disease comprising at least one natural extract selected from the group consisting of extract as an active ingredient am. The anti-carcinogenic and cancer metastasis inhibitory activity of Candelabra horseradish extract is known, such as [apoptosis inducer] in Japanese patent JP-0072888, [carcinostatic agent] in JP-0188530, and [agent for mitigating symptoms of cancer] in JP-0025933, JP -0019014 [skin-beautifying cosmetic] and JP-0342068 [moisturizing plant extract, and external preparation, cosmetic, bathing agent and detergent each containing the extract] are known to have therapeutic effects on skin diseases. In addition, [remedy for periodontosis] in JP-0122221, [coating composition for fry] in JP-0168429, [antipyorrheal composition] in JP-0184022, and [external preparation for skin] in JP-0008536 are disclosed, and Japanese Patent Laid-Open Patent Publication [immature dendritic cell-activating agent and use thereof] in JP-0238559 and [method for germination of seed of cimicifuga simplex group plant] in JP-0219461 are disclosed. However, to date, there is no known patent for the strong antithrombotic activity of the underground extract of Chotdae horseback riding.

KR 10-1303693 B

Hyang-Soon Cho, 2007. Master's thesis at Chung-Ang University. Antioxidant activity and inhibition activity of Nitric Oxide production in the underground part of Candlestick Horseback riding

The present invention has been devised to solve the problems of the prior art as described above, and the problem to be solved in the present invention is to provide a pharmaceutical composition for the prevention or treatment of thrombosis and a health functional food containing an extract of the underground part of Candlestick horseradish as an active ingredient. that you want to provide.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing an extract of the subterranean part of Candlestick horseback riding ( Cimicifuga simplex ) as an active ingredient.

It is preferable that the extract of the underground part of the Choddae horseback riding is an ethanol extract of the root in the shade.

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis comprising the extract of the chotdae horse riding ( Cimicifuga simplex ) underground.

It is preferable that the extract of the underground part of the Choddae horseback riding is an ethanol extract of the root in the shade.

The thrombosis prevention or treatment pharmaceutical composition and health functional food extract as an active ingredient of the present invention inhibits platelet aggregation, which plays a role in initiating thrombus formation along with inhibition of thrombus formation-related enzymes and blood coagulation factors. It exhibits strong antithrombotic activity by It is expected to be used for the prevention and treatment of thrombosis such as hemorrhagic stroke, and the active ingredient is processed into various forms such as extracts, powders, pills, tablets, etc. It is a very useful invention in the pharmaceutical and food industries.

1 is a photograph of the underground part of the Candlestick Horseback Riding.
Figure 2 shows the human platelet aggregation inhibitory activity of the ethanol extract of Choddae horseback riding: 1: solvent control (DMSO), 2: aspirin (0.25 mg/ml), 3: Chotdae horseback riding underground ethanol extract (0.25 mg/ml).

Hereinafter, the present invention will be described in detail.

In order to test the antithrombotic efficacy of the Candlestick equestrian basement, the inventors of the present invention prepared the Candlestick equestrian underground extract by a certain method, evaluated their antithrombotic activity, and recovered the Candlestick equestrian underground extract as an active ingredient, By confirming that the extract has excellent thermal stability and acid stability, the extract was intended to be used as a pharmaceutical composition and health functional food for preventing or treating/improving thrombosis.

Specifically, the present inventors use the underground part of Candelabra Horse which is known to be effective in various diseases such as measles, sore throat, lip inflammation and gingivitis, diarrhea, dysentery, etc. In order to develop, an ethanol extract was prepared for the underground part of Candlestick horseback riding, and their antithrombotic activity was directly inhibited by thrombin (Thrombin Time: TT), prothrombin time: PT, and active part on human plasma and human thrombin. By evaluating thromboplastin time (activated Partial Thromboplastin Time: aPTT), it was confirmed that the underground extract had very strong anticoagulant activity and strong platelet aggregation inhibitory activity.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis, which contains an extract of the underground part of the Candlestick horseback riding ( Cimicifuga simplex ) as an active ingredient.

It is preferable that the extract of the underground part of the Choddae horseback riding is an ethanol extract of the root in the shade.

In addition, the present invention provides a health functional food for the prevention or improvement of thrombosis comprising the extract of the chotdae horse riding ( Cimicifuga simplex ) underground.

It is preferable that the extract of the underground part of the Choddae horseback riding is an ethanol extract of the root in the shade.

Hereinafter, the production method and efficacy experiments of the extract of the Choddae horseback riding of the present invention will be described in more detail.

The present invention comprises the steps of preparing an extract from the underground part of Chotdae horseback riding; Evaluating the antithrombotic activity of the extract; and a step of examining the stability of the active substance.

"Chortdae equestrian underground extract" contained in the composition of the present invention can be obtained by extracting the dried root of Choddae equestrian horseradish with an organic solvent and filtering the extract using a filtration network of 0.06 mm or less, and concentrating it under reduced pressure. there is.

The drying may be preferably performed in the shade.

The organic solvent used in the present invention is water (cold water, hot water), alcohol, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), a mixed solvent of the lower alcohol and water, etc. can be used, and hot water or 95% ethanol extraction is most preferred.

As a preferred embodiment, the extract of the underground part of the Choddae riding horse of the present invention can be used by extracting the root of the Chotdae riding horse shade with hot water or ethanol. Here, the hot water or ethanol extract may be fractionated sequentially or separately with an organic solvent of hexene, ethyl acetate and butanol to additionally obtain a hexene fraction, an ethyl acetate fraction and a butanol fraction and a water residue.

In the present invention, as a result of measuring thrombin time, prothrombin time, and AP time with the extract of Candelabra horseshoe extract at a concentration of 5 mg/ml, the coagulation time is 8.02 times, 1.46 times, and 2.03 times longer than that of the non-additive group, respectively. As a result of measuring thrombin time, prothrombin time, and AP time at a concentration of 6 mg/ml, the coagulation time was 15 times longer, 1.62 times, and 2.87 times longer, respectively, compared to the non-additive treatment, which was concentration-dependent. It was confirmed that phosphorus antithrombotic activity was exhibited. In addition, as a result of confirming platelet aggregation inhibitory activity by adjusting the extract of Choddae horseback riding to 0.25 mg/ml concentration, it showed a 62.3% aggregation rate compared to the non-additive group, confirming that it had strong platelet aggregation inhibitory activity. These results suggest that the extract of Chotdae horseback riding horseradish exhibits very strong antithrombotic activity, and it can be substituted for anticoagulants and antiplatelet agents, such as aspirin, which have a high risk of side effects.

The underground extract of Candelabra horseradish of the present invention may be prepared as a powder through a conventional powdering process such as drying under reduced pressure and freeze drying, or spray drying. They are not degraded by various degrading enzymes in plasma, and they maintain activity even at 100°C heat treatment and at pH 2 in the human stomach.

The active ingredient of the present invention can be used for prevention or treatment of various diseases related to thrombosis. The diseases include, for example, arterial thrombosis, acute myocardial infarction, chest pain, dyspnea, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality change, blurred vision, epileptic seizures, pulmonary thrombosis , deep vein thrombosis, lower extremity edema, pain and acute peripheral arterial occlusion, and the like, and examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral vein sinus thrombosis, and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention can be prepared according to a conventional method according to the purpose of use. Oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injections of sterile injection solutions It can be formulated and used in various forms, such as oral administration, or administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

The pharmaceutical composition may additionally include a carrier, excipient or diluent, and the like, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil and the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

In a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, It is formulated by mixing sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may be used.

As a preferred embodiment, the oral liquid formulation may include suspensions, solutions, emulsions, syrups, etc., and various excipients, for example, wetting agents, sweetening agents, in addition to water and liquid paraffin, which are commonly used simple diluents, Perfumes, preservatives, and the like may be included.

As a preferred embodiment, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents, suppositories, etc. can be exemplified as formulations for parenteral administration. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.

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 with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient; Sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight daily Alternatively, it may be administered every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, disease severity, sex, weight, age, etc., the dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to a subject through various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

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 oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

In the present invention, the "subject" is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. and preferably a mammal, more preferably a human.

In addition, the health functional food of the present invention can be used in various ways, such as food and beverage effective for preventing or improving thrombosis. Foods containing the active ingredient of the present invention include, for example, various foods, beverages, gums, tea, vitamin complexes, health supplements, and the like, and may be used in powder, granule, tablet, capsule or beverage form. .

In general, the active ingredient of the present invention may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition may be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

The health functional food of the present invention may contain, as an additional ingredient, in addition to containing the above compound as an essential ingredient in the indicated ratio, a food pharmaceutically acceptable food supplement additive, such as natural carbohydrate and various flavoring agents.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and common sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

The flavoring agent includes: thaumatin; A natural flavoring agent such as stevia, such as rebaudioside A or glycyrrhizin, and a synthetic flavoring agent such as saccharin or aspartame may be used. 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 includes various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloids It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice, and fruit for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratio of these 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 through examples. The following examples are only preferred 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 extracts from the underground part of Chotdae horseback riding and analysis of their useful components

After purchasing the underground part of the Candlestick Horseback riding harvested in Gangwon-do in 2018, it was dried in the shade for one week and then used to prepare the ethanol extract. Specifically, 10 times of ethanol was added to the dry candlestick equestrian underground sample, extracted twice at room temperature, the extract was collected, filtered, and concentrated under reduced pressure to prepare a powder to prepare an ethanol extract.

Total polyphenols, total flavonoids, total sugar and reducing sugar contents were measured by component analysis of the extract of Chotdae horseback riding horse. For the total polyphenol content, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample, left at room temperature for 1 hour, and absorbance was measured at 725 nm. As a standard reagent, tannic acid was used. For the total flavonoid content, each sample was extracted with methanol for 18 hours, and 4 ml of 90% diethylene glycol was added to 400 μl of the filtered extraction sample, 40 μl of 1 N NaOH was added again, and the absorbance was measured at 420 nm after reaction at 37° C. for 1 hour. As a standard reagent, rutin was used. Total sugar was quantified using the phenol-sulfuric acid method, and sucrose was used as a standard reagent.

[Table 1] Extraction efficiency and useful component analysis of Chotdae horseback riding underground extract

As shown in Table 1, the extraction efficiency of the underground part of Candlestick horseback riding was 0.73%, which was lower than that of the general plant underground extract. The total polyphenol content was found to be high as 63.2 mg/g, and the total flavonoid content was found to be 24.4 mg/g. The total sugar content was 162.8 mg/g, which was lower than that of other plant root extracts. This means that the underground part of Candlestick Horseback riding is mostly composed of fiber.

Example 2: Evaluation of blood clotting inhibitory activity of Chotdae horseback riding underground extract

The blood coagulation inhibitory activity of the ethanol extract of Example 1 was evaluated, and the results are shown in Table 2. At this time, the blood coagulation inhibitory activity evaluation method of Chotdae horseback 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 AP time were measured. For plasma, commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China) was used, and thrombin time, prothrombin time, and AP time were measured as follows.

Thrombin Time

At 37°C, 50 μl of 0.5U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ , and 10 μl of sample extracts of various concentrations were mixed in a tube of Amelung coagulometer KC-1A (Japan) and reacted for 2 minutes, and then 100 μl of plasma was collected. After addition, the time until plasma coagulation was measured. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. DMSO showed a coagulation time of 32.1 seconds. The thrombin inhibitory effect was expressed as the average of three or more repeated experiments, and the thrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of sample solutions of various concentrations were added to the tube of Amelung coagulometer KC-1A (Japan), heated at 37° C. for 3 minutes, 130 μl of PT reagent was added, and plasma was coagulated. The time until completion was expressed as the average value of the experiment repeated three times. Aspirin (Sigma Co., USA) was used as a control, and DMSO was used instead of the sample as a solvent control. DMSO showed a clotting time of 18.1 seconds. The prothrombin inhibitory activity was expressed as the value obtained by dividing the coagulation time at the time of sample addition by the coagulation time of the solvent control.

Activated Partial Thromboplastin Time (aPTT)

100 μl of plasma and 10 μl of sample extracts of various concentrations were added to the tube of ^Amelung coagulometer KC-1A (Japan) and heated at 37°C for 3 minutes. Incubated for 3 minutes. Then, after adding 50 μl CaCl ₂ (35 mM), the time until plasma coagulation was measured. As a solvent control, DMSO was used instead of the sample, and in this case, the coagulation time was 55.1 seconds. The results of aPTT were expressed as the average value of the experiment repeated three times, and the blood coagulation factor inhibitory activity was expressed as the value obtained by dividing aPTT at the time of sample addition by the aPTT of the solvent control.

[Table 2] Blood clotting inhibitory activity of Chotdae horseback riding underground extract

As a result, as a result of measuring thrombin time, prothrombin time, and AP time with the extract of Choddae horseback riding at a concentration of 5 mg/ml, the coagulation time was extended 8.02 times, 1.46 times, and 2.03 times, respectively, compared to the non-additive group, showing strong thrombosis At the concentration of 6 mg/ml, the coagulation time was 15 times longer, 1.62 times, and 2.87 times longer, respectively, compared to the no-addition group, and at the concentration of 7 mg/ml, the coagulation time was 15 times or more, respectively, compared to the no-addition group, The clotting time was extended by 2.04 times and 3.71 times, indicating strong concentration-dependent antithrombotic activity. These results, considering that aspirin, which is currently used as an antithrombotic agent in clinical practice, prolongs thrombin time, prothrombin time, and AP time by 1.56 times, 1.50 times, and 1.56 times, respectively, at a concentration of 1.5 mg/ml compared to the non-additive group. It is suggested that the underground extract can replace anticoagulants and antiplatelet drugs such as aspirin, which are highly concerned about side effects.

Example 3: Platelet aggregation inhibitory activity of Chotdae horseback riding underground extract

The human platelet aggregation inhibitory activity of the Choddae horseback riding underground extract of Example 1 was evaluated, and the results are shown in Table 3 and FIG. 2 . Platelets are disk-shaped small cells that circulate in blood vessels together with various blood cells. Instead of having a nucleus, they have cytoplasmic granules containing various substances related to vascular damage protection and platelet aggregation in high concentrations. It is an important cell that secretes aggregation factors and forms a primary hemostatic plug by binding to collagen exposed due to endothelial cell damage and initiating thrombus formation. Therefore, platelet aggregation inhibition is a very important activity to prevent thrombus formation. The platelet aggregation inhibitory activity was evaluated according to the following method.

Platelet aggregation inhibition activity

Platelets were human concentrated platelets, which were 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). Then, re-suspended in suspending buffer (138mM NaCl, 2.7mM KCl, 12mM NaHCO ₃ , 0.36mM NaH ₂ PO ₄ , 5.5mM Glucose, 0.49mM MgCl ₂ , 0.25% gelatin, pH 7.4), and then at 3,000 rpm for 10 minutes After centrifugation, it was re-suspended in suspending buffer, and the platelet count was adjusted to be 4x10 ⁹ /ml. Then, 2.5 μl collagen was added to 1 ml suspension, reacted for 5 minutes, and platelet aggregation was measured at 37° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 3] Platelet aggregation inhibitory activity of Chotdae horseback riding underground extract

As shown in Table 3, first, aspirin exhibited aggregation of 35.1% at a concentration of 0.25 mg/ml, thereby showing strong platelet aggregation inhibitory activity, thereby confirming the rationale for its use as an antithrombotic agent in clinical practice. On the other hand, as a result of confirming platelet aggregation inhibitory activity by adjusting the extract of Choddae horseback riding underground to 0.25 mg/ml concentration, it showed a 62.3% aggregation rate compared to the non-additive group, confirming that it had strong platelet aggregation inhibitory activity. Therefore, it was confirmed that the extract of Choddae horseback riding horses can be used as an antithrombotic agent due to its strong platelet aggregation inhibitory activity.

Example 4: Evaluation of Plasma, Acid and Thermal Stability of Chotdaeseungae Underground Extract

Plasma stability, thermal stability and acid stability with respect to antibacterial activity were confirmed for the extract of Chotdae horseback riding underground part obtained in Example 1. As a result, the active extract showed no significant loss of antithrombotic activity even when heat treated at 100° C. for 1 hour, treated at pH 2 (0.01M HCl) for 1 hour, and treated in plasma for 1 hour, indicating high stability. Therefore, it is expected that the extract of the Choddae horseback riding underground part will maintain excellent antithrombotic activity even under various food processing processes.

Claims (3)

Chotdae riding horse ( Cimicifuga simplex ) A pharmaceutical composition for preventing or treating thrombosis containing the underground extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extract of the Choddae Equestrian horse is an ethanol extract of the root in the shade. A health functional food for preventing or improving thrombosis comprising the active ingredient according to claim 1 or 2.

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