KR20220005165A - Pharmaceutical composition comprising the extract of rose flower 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 rose flower 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 dried rose flower as an active ingredient for preventing or treating/ameliorating thrombosis through inhibited blood coagulation. According to the present invention, the extract of rose flower, which is an effective component of the pharmaceutical composition and health functional food for preventing or treating thrombosis, shows a strong antithrombotic activity through inhibited thrombogenesis-related enzymes and blood coagulation factors, 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 extract 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 preventing or treating thrombosis containing rose flower extract 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 a rose flower extract as an active ingredient, and more specifically, to a dried rose flower extract as an active ingredient. It relates to a pharmaceutical composition for preventing or treating/improving thrombosis through inhibition of blood coagulation and a functional food.

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. have. 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. Accordingly, 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.

Rose is a perennial woody plant belonging to the genus Rosa of the family Rosaceae, and more than 100 species are known worldwide. A common rose is a horticultural species (Rosa hybrida Hort.), and flowers (roseflowers) and fruits (rosehips) have been used medicinally and edible. In particular, rose flowers have been used for decoration and fragrance due to their beautiful appearance and unique fragrance, and rose oil has been used in folklore to relieve inflammation and treat atopy.

On the other hand, research on roses has been focused on improving rose flower varieties and analyzing fragrance components, but recently, useful physiological activity studies of rose flower extracts are also being conducted. The reported physiological activities of roses include anti-inflammatory activity (Sunmi Lee et al., 2015, Korean J. Food Nutr. 28: 551-557), and antioxidant activity (Cai YZ et al., 2005, J Agric Food Chem 53:9940-9948; Tateyama CG et al., 1997. Nippon Shokuhin Kogyo Gakkaishi 44:640-646; Chun HK et al., 2004. Korean J. Commun. Living Sci. 15: 67-76), anti-atopic activity (Jeon JH et al., 2009. Arch Pharm Res 32: 823-830), collagenase (clloagenase) and elastinase (elastase) inhibitory activity (Thring TSA, 2009. BMC Complement Altern Med 9:27-38), ACE inhibitory activity, HMG-CoA reductase inhibition Activity and thrombolytic activity (Kwon Eun-kyung et al. 2006. Korean Journal of Food Science 38: 691-698), etc. are known, and antibacterial activity against Salmonella enteritidis (Yujin Song, 2009. Korean J. Microbiol. 45, 318-323) , Antibacterial activity against Helicobacter pylori (Yang Yunhee, 2012, Chungbuk National University master's thesis), antibacterial activity against various skin bacteria (Park D, et al., 2016. Regul. Toxicol. Pharmacol. 76: 57-62) and chinese hamster ovary cells Growth promoting activity (Cho YS et al., 2007. J. Korean Soc. Appl. Biol. Chem. 50: 132-135), growth promoting effect of 3T3-L1 cells (Chun HK et al., 2004. Korean J. Commun. Living Sci 15: 67-76) are known. In addition, rose component (Cho YS et al. 2007. J. Korean Soc. Appl. Biol. Chem. 50: 132-135) and pigment safety (Yang M et al., 2002. J. Korean Soc. Food Sci. Nutr. 31: 539-542) has also been reported. However, there have been no reports of strong antithrombotic activity of rose extracts to date.

Patents related to roses include [Rose petal extract having skin whitening function and skin whitening cosmetic composition containing the same] in Korean Patent Registration No. 10-1989999 [Rose ripening extract and manufacturing method], and [Rose petal extract having skin whitening function] in No. 10-0389096 , [COSMETIC COMPOSITION CONTAINING fermented black yeast broth of rose petals of Provence] in No. 10-2100820, [Rose ripening extract and manufacturing method thereof] in No. 10-1989999, [Saccharification extract and rose flower extract in No. 10-1975951 Most of cosmetic related patents such as manufacturing method and whitening cosmetic composition containing extract prepared by the above method] ) Manufacturing method and composition thereof], [Production method of rose tea] in No. 10-1953718, [Strawberry jam containing onion and rose and production method thereof] in No. 10-2056473-[ Manufacturing method of beer containing rose flower extract], in No. 10-0868580 [Method for preparing flower wine using rose flower and its extract], in No. 10-0755190 [Hot water extract of rose petals and manufacturing method of functional beverage using the same] , [Method for manufacturing stamen using rose flowers and extracts thereof] in No. 10-0868580, [Method for manufacturing ice cream mainly made of roses] in No. 10-0439567, A number of food-related patents such as [Method for manufacturing flower wine using the extract] have been disclosed. In addition, in No. 10-1870780 [Women's cleaning composition comprising damask rose oil and propolis extract as active ingredients], No. 10-1394550 [Antibacterial or antibacterial composition containing rose flower extract as an active ingredient] Inflammatory composition], antibacterial and anti-inflammatory related patents such as [composition for prevention and treatment of atopic dermatitis] in No. 10-1075006, and [composition for treatment of ischemic brain disease containing rose flower extract as an active ingredient in No. 10-1384351 ] is known. In this case, the composition for the treatment of ischemic brain disease reduces the size of the cerebral infarct region and cerebral edema in ischemic brain injury induced by middle cerebral artery occlusion and reperfusion, lowers the level of lipid peroxidation and brain inflammation, and significantly improves brain damage and behavioral disorders. It is disclosed to exhibit brain protective activity. Meanwhile, as published patents, [Method for manufacturing granular tea from edible rose petals] in No. 10-2011-0009267, [Composition comprising white rose flower extract as an active ingredient] in No. 10-2011-0072153, No. 10-2015- [Rose extract using rose petals, rose red pepper paste, rose enzyme syrup, rose snack, and nutritional food composition method and composition using various petals] in No. 0012720, [Rose flower extract or its composition] in No. 10-2020-0037537 Composition for the prevention or treatment of benign prostatic hyperplasia containing fraction], [Composition for improvement of hair loss and white hair containing extract of red rose petals] is disclosed in No. 10-2019-0137255. However, there is no known patent related to the antithrombotic activity of rose flower extract to date.

KR 10-1384351 B

Kwon Eun-kyung et al. 2006. Journal of the Korean Society for Food Science 38: 691-698

The present invention has been devised to solve the problems of the prior art as described above, and an object to be solved in the present invention is to provide a pharmaceutical composition for preventing or treating thrombosis containing a rose flower extract as an active ingredient, and a health functional food would like to

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing a rose flower extract as an active ingredient.

The rose flower extract is preferably a methanol extract of dried rose flower.

In addition, the present invention provides a health functional food for preventing or improving thrombosis comprising a rose flower extract.

In addition, the present invention provides an anticoagulant containing a rose flower extract as an active ingredient.

The rose flower extract as an active ingredient of the pharmaceutical composition for preventing or treating thrombosis of the present invention and a health functional food exhibits strong antithrombotic activity through inhibition of thrombus formation-related enzymes and blood coagulation factors, and at the same time, hemolysis against human red blood cells No activity, excellent thermal stability, and no loss of blood coagulation factor inhibitory effect and thrombus formation-related enzyme inhibitory effect even under acidic conditions of pH 2 and plasma. It is expected that it can be used for the prevention and treatment of This is a very useful invention.

1 shows a dried rose flower.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention prepared a rose flower extract by a certain method in order to test the antithrombotic effect on rose flowers, and as a result of evaluating its antithrombotic activity, a strong anticoagulant component was recovered from the rose flower extract, By confirming that the extract has no human red blood cell hemolytic activity, and has excellent thermal stability and acid stability, the extract is used for preventing or treating/improving thrombosis, a pharmaceutical composition and a health functional food, more specifically, blood clotting inhibitory activity It was intended to be used as an anticoagulant representing

Specifically, the present inventors are effective in skin diseases and inflammation in the folklore, using roses known to have excellent antibacterial and antioxidant activity to develop pharmaceutical compositions and health functional foods for preventing or treating/improving thrombosis, dried A methanol extract was prepared from the rose flower, and its antithrombotic activity was evaluated for Thrombin Time, Prothrombin Time, and Activated Partial Thromboplastin Time on human plasma and human thrombin: aPTT), it was confirmed that the extract showed very strong thrombin inhibition and anticoagulant activity through blood clotting factor inhibition.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating thrombosis containing a rose flower extract as an active ingredient.

The rose flower extract is preferably a methanol extract of dried rose flower.

In addition, the present invention provides a health functional food for preventing or improving thrombosis comprising a rose flower extract.

The rose flower extract is preferably a methanol extract of dried rose flower.

In addition, the present invention provides an anticoagulant containing a rose flower extract as an active ingredient.

The rose flower extract is preferably a methanol extract of dried rose flower.

Hereinafter, a method for preparing a rose flower extract of the present invention and an efficacy test will be described in more detail.

The present invention comprises the steps of drying a rose flower; preparing an extract from dried rose flowers; Evaluating the antithrombotic activity of the extract; and a step of examining the stability of the active substance.

"Rose flower extract" contained in the composition of the present invention is a step of drying mature flowers of roses, extracting them with an organic solvent, filtering the obtained extract using a filtration network of 0.06 mm or less, and concentrating it under reduced pressure. can be obtained by

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, 80% methanol, 95% ethanol extraction is most preferred.

In a preferred embodiment, the rose flower extract of the present invention may be used after drying the mature rose flower and then extracting it with hot water, methanol or ethanol. Here, the obtained extract may be sequentially or individually fractionated 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 by using the rose flower extract at a concentration of 5 mg/ml, strong anticoagulant activity was confirmed, which was extended by more than 15 times compared to the non-additive group. This activity was stronger than the anticoagulant activity of aspirin (1.5mg/ml), which is known as an antithrombotic agent.

The rose flower extract 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 further 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 hot water and methanol extracts of rose flowers and review of physicochemical properties of these extracts

Dried rose flowers (in the form of flower tea) sold in Korea were purchased and used to prepare hot water and methanol extracts. A photograph of a dried rose flower is shown in FIG. 1 . In the case of hot water extract, 20 times distilled water was added to the dried rose flower sample and extracted for 5 minutes at 100 ° C. The methanol extract was extracted twice at room temperature after adding 20 times 80% methanol to the dried rose flower sample. did The extracts were collected, filtered, and concentrated under reduced pressure to prepare a powder, respectively, to prepare hot water and methanol extracts. The pH, brix and acidity of rose flowers were evaluated for the hot water extract, and the results are shown in Table 1.

[Table 1] pH, brix and acidity of rose flowers

The dried rose flower had a pH of 4.2, a brix of 32, and an acidity of 6.08.

Meanwhile, the results of evaluating the color difference between the dried rose flower hot water extract and the methanol extract are shown in Table 2. The brightness of the hot water extract was 8.40, the degree of redness was 2.53, the degree of yellowness was 1.86, and the color difference was 84.09. The brightness of the methanol extract was 9.82, the degree of redness was 0.16, the degree of yellowness was 3.47, and the color difference was 82.63.

[Table 2] Color difference analysis of rose flower hot water extract and methanol extract

Example 2: Extraction efficiency and component analysis of methanol extract of rose flower

The components of the methanol extract of rose flower prepared in Example 1 were evaluated, and the results are shown in Table 3. At this time, for the total polyphenol content of the rose flower extract, 50 μl of Folin-ciocalteau and 100 μl of Na ₂ CO ₃ saturated solution were added to 400 μl of the extraction sample solution, and the absorbance was measured at 725 nm after standing at room temperature for 1 hour. 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.

[Table 3] Extraction efficiency and useful component analysis of methanol extract of rose flower

As shown in Table 3, the extraction efficiency of the rose flower extract was 24.1%, the total polyphenol content was 59.2 mg/g, the total flavonoid content was 47.7 mg/g, and the total sugar content was 88.6 mg/g.

Example 3: Evaluation of blood clotting inhibitory activity of rose flower extract

The blood clotting inhibitory activity of the methanol extract of rose flower of Example 1 was evaluated, and the results are shown in Table 4. At this time, the blood coagulation inhibitory activity of the rose flower 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), heated at 37° C. for 3 minutes, and then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added, and 3 Incubated for minutes. After 50 μl CaCl ₂ (35mM) was added, the time until plasma coagulated 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 result of aPTT was 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 the aPTT at the time of sample addition by the aPTT of the solvent control.

[Table 4] Blood clotting inhibitory activity of rose flower extract

As shown in Table 4, the rose flower extract showed strong thrombin inhibition, which was prolonged more than 15-fold at the concentration of 5 mg/ml. In the case of AP time, which indicates blood clotting factor inhibition, the rose flower extract showed prothrombin time and AP time that were 15 times or more prolonged at a concentration of 5 mg/ml. These results show that rose flower extract can be developed as an antithrombotic agent, especially an anticoagulant.

Example 4: Human hemolytic activity of rose flower extract

As rose flowers were used as tea, the toxicity of methanol extract of rose flowers was judged to be low. Therefore, in order to evaluate the acute toxicity potential of the methanol extract of rose flower, the hemolytic activity of human red blood cells was evaluated, and the results are shown in Table 5. At this time, hemolytic activity was evaluated according to an existing report (Ho-Yong Son, 2014 ㆍKorean J. Microbiol. Biotechnol. 42: 285-292), and 100 μl of human red blood cells washed three times with PBS was simply placed in a 96-well microplate. 100 μl of sample solutions of various concentrations were added, followed by reaction at 37° C. for 30 minutes. After that, the reaction solution was centrifuged (1,500 rpm) for 10 minutes, and 100 μl of the supernatant was transferred to a new microtiter plate. was measured in DMSO (2%) was used as a solvent control of the sample, and Triton X-100 (1mg/ml) was used as an experimental control for hemolysis of red blood cells. Hemolytic activity was calculated using the following formula.

[Table 5] Human hemolytic activity of rose flower extract

First, DMSO and water used as controls had no hemolytic activity, and it was confirmed that triton X-100 hemolyzed red blood cells 100% at a concentration of 1 mg/ml. In addition, in the case of amphotericin B, which is used as an anticancer agent and antifungal agent, it was confirmed that more than 50% of red blood cells were hemolyzed at a concentration of 0.025 mg/ml. The rose flower extract of the present invention did not show red blood cell hemolytic activity up to a concentration of 1 mg/ml.

Example 5: Evaluation of Plasma, Acid and Thermal Stability of Rose Flower Extract

Plasma stability, thermal stability and acid stability for antithrombotic activity were confirmed for the methanol extract of rose flower obtained in Example 1. The methanol extract of rose flower did not show any decrease in blood clotting inhibitory activity even after 1 hour heat treatment at 100° C., 1 hour treatment at pH 2 (0.01M HCl), and 1 hour treatment in plasma. Therefore, it is expected that the methanol extract of rose flower contains antithrombotic active substances of various components with acid resistance and heat resistance.

Claims (4)

A pharmaceutical composition for preventing or treating thrombosis containing a rose flower extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the rose flower extract is a methanol extract of dried rose flower. A dietary supplement for preventing or improving thrombosis, comprising rose flower extract. An anticoagulant containing rose flower extract as an active ingredient.

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