KR20200005774A - Pharmaceutical composition comprising the butanol fraction obtained from ethanol extract of apis mellifera 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 an antithrombotic composition containing a butanol fraction of an apis mellifera ethanol extract as an active ingredient, and more particularly, to a pharmaceutical composition and a functional health food for preventing or treating thrombosis through blood coagulation inhibition, which prepares an ethanol extract of an imago of western apis mellifera used for apiculture, and sequentially fractionates the same with an organic solvent to contain a recovered butanol fraction as an active ingredient. According to an embodiment of the butanol fraction of an apis mellifera ethanol extract as an active ingredient of the pharmaceutical composition and the functional health food for preventing or treating thrombosis exhibits strong antithrombotic activity by thrombogenesis-related enzyme inhibition and blood coagulation factor inhibition, does not exhibit aggregation activity against human platelets, has excellent thermal stability, and does not exhibit loss of an effect of thrombogenesis-related enzyme and blood coagulation factor inhibition even under acidic condition of pH 2 and plasma, thereby expecting to be used for preventing and treating thrombosis such as ischemic stroke and hemorrhagic stroke through improvement of blood circulation. Also, the active ingredient has an excellent effect of being processed in various forms such as extract, powder, pill, tablet, etc. to be prepared in a form capable of constant administration, thereby being very useful in a pharmaceutical industry and a food industry.

Description

PHARMACEUTICAL COMPOSITION COMPRISING THE BUTANOL FRACTION OBTAINED FROM ETHANOL EXTRACT OF APIS MELLIFERA AS AN EFFECTIVE COMPONENT FOR PREVENTION OR TREATMENT OF THROMBOSIS AND HEALTH FUNCTIONAL FOOD COMPRISING THE SAME}

The present invention relates to an antithrombotic composition containing butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient. It relates to a pharmaceutical composition for the prevention or treatment / improvement of the thrombosis through the blood coagulation inhibition containing butanol fraction recovered as an active ingredient and a health functional food.

Blood as a human component has various important functions such as transporting and buffering oxygen, nutrients, and wastes, maintaining body temperature, controlling osmotic pressure and ion balance, maintaining moisture, controlling fluid, maintaining and regulating blood pressure, and defending the body. have. Normal blood circulation facilitates blood circulation as the blood coagulation reaction system and the thrombolysis reaction system in the body complement each other, and among them, the mechanism of the blood coagulation reaction system forms platelet thrombus by adhering and agglomerating platelets to blood vessel walls. In addition, it has been reported that the blood coagulation system is activated to form fibrin clots around platelet aggregates.

On the other hand, the generation of fibrin thrombus is a multi-step reaction of numerous blood coagulation factors to activate the thrombin involved in fibrin coagulation, which finally produces fibrin monomers from fibrinogen, and the fibrin monomers are polymerized by calcium, which causes platelets and endothelium. It binds to cells and creates permanent thrombi, forming fibrin polymers cross-linked by factor XIII. In addition, thrombin plays a pivotal role in thrombus formation by activating platelets, factor V and factor VII to promote blood coagulation. Therefore, thrombin activity inhibitors can be used as a prophylactic and therapeutic agent that is very useful for various thrombotic diseases that occur due to excessive blood clotting. On the other hand, in the endogenous thrombus generation pathway, sequential activation of factor XII, factor XI, factor XII, factor IX and factor X is followed by activation of prothrombin to finally activate thrombin. It has been a development target for the treatment of sexual diseases, and in the case of exogenous thrombus generation pathway, thrombus generation by activation of factor II (prothrombin), factor V, factor VII, and factor X is known. Until now, various anticoagulants such as heparin, coumarin, aspirin, urokinase, antiplatelet agents, thrombolytic agents, etc. have been used for the prevention and treatment of thrombotic diseases, but they are very expensive and have hemorrhagic side effects, gastrointestinal disorders and hypersensitivity. The use is limited by reaction etc.

On the other hand, bees ( Apis mellifera ) are insects that live together with logging bees and are the oldest insects bred to mankind. It is a beneficial insect that plays a decisive role in the planting of pollen of plants all over the world, and also produces honey, honeycomb, royal jelly, propolis, pollen, bee venom, etc. through beekeeping. Bees are one of the most edible insects in the world, and they are also used for medicinal purposes.

Recently, studies on the high value of insects have been conducted by changing the social perception of edible insects and activating the insect industry, and in the case of bees, the antioxidant and antidiabetic activities of hydrothermal extracts (Melo da Cunha JDS et al., 2018.PLoS One.13 (6): e0197071), antibacterial activity of bee venom (Somwongin S. et al., 2018.Toxicon. 145: 32-39; Han Sangmi et al., 2011. Journal of Fish Pathology 24: 113-120), anticoagulant activity of bee venom (H. Zolfagharian et al., 2015. J. Pharmacopuncture, 18 (4): 7-11), lytic activity of fibrin blood clots of bee larvae extract (Hyun-ae Kim, 2013, Journal of Sericultural and Entomological Science v. 51 no.2, pp. 147-152), various enzyme inhibitors from bee venom (Tae-Sook Jung et al., 1997. Korean Journal of Apiculture 12: 85-92; However, the antithrombotic activity of bee extracts other than bee venom has not been known to date.

Honey related patents are mostly related to bee beekeeping related devices such as [beekeeping control system] (Korean Patent No. 10-1867985), [vehicle beehive multi-stage storage device] (Korean Patent No. 10-1856641), Recently, [compositions for the prevention and treatment of bee infectious diseases] (Korean Patent No. 10-1847532), [compositions for the prevention and treatment of buzzer diseases] (Korean Patent No. 10-1828563), [Immune for honeybees Compositions] (Patent Registration No. 10-1424083), [Apiculture Veterinary Composition] (Republic of Korea Patent Registration No. 10-1185328) Patents related to bee beekeeping is disclosed. In particular, most of the useful functional patents related to bees are concentrated on bee venom, and the Republic of Korea Patent No. 10-1316600 is a fish feed composition containing bee venom, and the Republic of Korea Patent No. 10-1834758 is effective for bee venom. A composition for preventing or treating periodontitis, which is included as a component, is disclosed, and in Korean Patent Publication No. 10-2009-0114999, [Pharmaceutical composition for treating cirrhosis including bee venom], No. 10-2012-0047593 Composition for inhibiting breast cancer metastasis comprising the same as an active ingredient is known. However, the antithrombotic activity of bee extracts is not known to date.

 Poster presented at the 58th annual general meeting of the Korean Society of Life Science and International Conference 2017 P2-31.  Poster presented at the 58th annual general meeting of the Korean Society of Life Science and International Conference 2017 P2-33.  H. Zolfagharian et al., 2015. J. Pharmacopuncture, 18 (4): 7-11: Anticoagulant Activity of Bee Venom.  Kim, Hyun-ae et al., 2013, Journal of Sericultural and Entomological Science, v.51, no.2, pp.147-152: Soluble Activity of Fibrin Thrombosis of Bee Larva Extract.

The present invention has been made to solve the problems of the prior art as described above, the problem to be solved in the present invention is to provide an antithrombotic composition containing butanol fraction of bee ethanol extract as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient.

The present invention also provides a blood coagulation inhibitor containing butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient.

The present invention also provides a health functional food for preventing or improving thrombosis containing a butanol fraction of a bee ( Apis mellifera ) ethanol extract as an active ingredient.

Butanol fraction of bee ethanol extract as an active ingredient of the pharmaceutical composition for the prevention or treatment of thrombosis of the present invention and the health functional food, as demonstrated through the examples of the present disclosure, It exhibits strong antithrombotic activity by inhibition, shows no coagulation activity on human platelets, has excellent thermal stability, and shows a loss of the inhibitory effect of blood clotting factor-related enzymes and coagulation factors even in acidic conditions and plasma of pH 2. Therefore, it is expected to be used for the prevention and treatment of thrombosis such as ischemic stroke and hemorrhagic stroke by improving blood circulation, and the active ingredient is processed into various forms such as extract, powder, pill, tablet and can be taken at all times. Pharmaceutical and food industries It will be very useful inventions.

1 is a photograph of bee larvae, pupa, adult powder used in the experiment.
Figure 2 shows the human platelet aggregation inhibitory activity of adult honeybee ethanol extract and sequential organic solvent fractions thereof. 1: solvent control (DMSO), 2: aspirin (0.50 mg / ml), 3: aspirin (0.25 mg / ml), 4: bee adult ethanol extract (0.25 mg / ml), 5: hexene fraction of bee adult ethanol extract (0.25 mg / ml), 6: ethyl acetate fraction of bee adult ethanol extract (0.25 mg / ml) 7: butanol fraction of bee adult ethanol extract (0.25 mg / ml), 8: organic solvent fraction of bee adult ethanol extract Water residue (0.25 mg / ml).

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The inventors of the present invention, in order to assay the antithrombotic efficacy in honeybees known for its excellent nutrition and various pharmacological activities, first purchased the larvae, pupa and adults of the bees, and then rapidly frozen at -20 ℃ to kill Next, lyophilized at -50 ℃, and then using a mortar and pestle was prepared bees larvae, pupa and adult powder, respectively. Thereafter, an ethanol extract was prepared using a manufactured powder, and the butanol fraction of the ethanol extract was recovered as an antithrombotic active ingredient, and the butanol fraction was confirmed to have excellent thermal stability and acid stability. Prophylactic or therapeutic / improvement pharmaceutical composition and health functional food.

Specifically, the inventors of the present invention target bee larvae, pupa and adult to develop pharmaceutical compositions and health functional foods for the prevention or treatment / improvement of thrombosis using bees known to have anti-inflammatory, antidiabetic and antioxidant effects. Ethanol extracts were prepared, and these extracts were subjected to thrombin time, prothrombin time, and activated partial thromboplastin time (aPTT) and platelet aggregation inhibitory activity against human thrombin. As a result, it was confirmed that only the bee adult extract did not show thrombogenic activity (hemostasis) such as platelet aggregation promotion. In fact, bee larva and pupa extract showed strong platelet aggregation activity, and it was confirmed that the thrombus promoting activity was stronger than the antithrombotic activity. Thus, after sequential organic solvent fractions were prepared for adult adult bee extracts, the antithrombotic activity of each bee was evaluated, and it was confirmed that butanol fraction of bee ethanol extract showed excellent anticoagulant activity without platelet aggregation promoting activity. In particular, bee butanol fractions showed anticoagulant activity in a concentration-dependent manner, and increased thrombin time, prothrombin time and apitime by 1.55 times, 1.39 times and 1.51 times, respectively, at 5 mg / ml. At 7 mg / ml concentrations, thrombin time, prothrombin time, and apitime were extended 2.01, 1.54, and 1.98 times, respectively, compared to no addition. In addition, the butanol fraction showed little hemolytic activity against human erythrocytes and did not cause acute toxicity.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombosis containing butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient.

The present invention also provides a blood coagulation inhibitor containing butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient.

The present invention also provides a health functional food for preventing or improving thrombosis containing a butanol fraction of a bee ( Apis mellifera ) ethanol extract as an active ingredient.

Hereinafter, the production method and efficacy test of the bee extract and the anti-thrombotic active fraction of the present invention will be described in more detail.

The inventors of the present invention, in order to achieve the object of the present invention, bee breeding step; Bee larvae, pupa, adult recovery / pretreatment and lyophilization steps; Powdering stages of bee larvae, pupa, adults; Preparation of extracts of bee larvae, pupa, adult; Preparation of sequential organic solvent fractions of hexene, ethyl acetate, butanol from honeybee adult extract and subsequent preparation of water residues; Experiments were performed to evaluate the antithrombotic activity of the extracts and fractions and to investigate the stability of the active substances of the butanol fraction.

In a preferred embodiment, the present invention, after raising bees in the same manner as conventional insect breeding, and recovering the larvae, pupa, and adults at the beekeeping site, and then rapidly frozen to -20 ℃ to kill, then -50 ℃ After freeze-drying in, using a mortar and pestle to make a powder and extracted with a solvent, the extract was filtered using a filter net of 0.06mm or less, concentrated under reduced pressure to prepare an extract, and then sequentially fractionated with an organic solvent and an organic solvent Fractions can be obtained.

The solvent used in the present invention includes water (cold water, hot water), spirits, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, alcohol, propanol, butanol, etc.), mixed solvents of the lower alcohols with water, and the like. Hydrothermal, or 95% ethanol extraction is most preferred.

As the organic solvent used in the present invention, hexene, ethyl acetate and butanol may be used, and extracts may be sequentially or separately fractionated with these organic solvents to obtain a hexene fraction, an ethyl acetate fraction, a butanol fraction and a water residue.

In the present invention, the beet larvae, pupa, adult ethanol extract was measured in the concentration of 5mg / ml, the thrombin time, prothrombin time, apititime was measured, 0.99 ~ 1.04 times, 0.99 ~ 1.04 times, respectively, compared to the non-added By extending 0.96 to 1.02 fold, it was confirmed that there was little activity of thrombin inhibition, prothrombin inhibition and coagulation factor inhibition. These results indicate that aspirin (trade name), which is commercially used as an antithrombotic agent, extends thrombin time, prothrombin time, and epitaxial time by 1.43 times, 1.73 times and 1.42 times, respectively, at 1.5 mg / ml. Is compared to However, the butanol fraction of the organic solvent fraction of bee ethanol extract showed excellent anticoagulant activity and extended thrombin time, prothrombin time and apitime by 1.55, 1.39 and 1.51 times, respectively, at 5 mg / ml. At 7 mg / ml concentration, thrombin time, prothrombin time, and apitime were extended by 2.01, 1.54, and 1.98 times, respectively, compared to the non-added groups, indicating the concentration-dependent strong anticoagulant activity. These results suggest that the butanol fraction of bee ethanol extract can be developed as an actual antithrombotic agent.

The butanol fraction of the bee ethanol extract of the present invention may be prepared into a powder through a conventional powdering process such as vacuum drying and freeze drying or spray drying. The butanol fraction of bee ethanol extract is not degraded to various enzymes in plasma, and is maintained at 100 ° C. heat treatment and pH 2 in the human stomach.

The active ingredient of the present invention can be applied as a medicinal use of an anticoagulant, that is, a blood coagulation inhibitor, and can be used for the prevention or treatment of various diseases related to thrombosis. The diseases are, for example, arterial thrombosis, acute myocardial infarction, chest pain, shortness of breath, loss of consciousness, ischemic stroke, hemorrhagic stroke, headache, dyskinesia, paresthesia, personality changes, decreased vision, epileptic seizures, pulmonary thrombosis , Deep vein thrombosis, lower extremity edema, pain, and acute peripheral arterial obstruction. Examples of venous thrombosis include deep vein thrombosis, portal vein thrombosis, acute renal vein occlusion, cerebral venous thrombosis, and central retinal vein occlusion.

The pharmaceutical composition comprising the active ingredient of the present invention may be orally formulated as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It may be formulated and used in various forms, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Such pharmaceutical compositions may further include carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include a filler, an anticoagulant, a lubricant, a humectant, a perfume, 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, which solid preparations comprise at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition, lubricants such as magnesium stearate, talc and the like may also be used in addition to simple excipients.

As a preferred embodiment, oral liquid preparations may be exemplified by suspending agents, liquid solutions, emulsions, syrups, and the like, and in addition to commonly used simple diluents such as water and liquid paraffin, various bee excipients such as wetting agents, sweetening agents, Fragrances, preservatives and the like.

As a preferred embodiment, the preparation for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories and the like. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, activity of the drug, Sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in 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 per kg of body weight, preferably 100 to 3,000 mg daily. Or every other day or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

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

As used herein, "administration" means providing a patient with any substance by any suitable method, wherein the route of administration of the pharmaceutical composition of the present invention is oral or parenteral via all common routes as long as the target tissue can be reached. Oral administration. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

"Subject" in the present invention 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 mammals, and more preferably humans.

In addition, the health functional food of the present invention can be used in a variety of foods and drinks effective for preventing or improving thrombosis. Examples of the food containing the active ingredient of the present invention include various foods, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, and can be used in the form of powders, granules, tablets, capsules, or beverages. .

The active ingredient of the present invention can generally be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g based on 100ml.

In addition to containing the compound as an essential ingredient in the indicated ratios, the health functional food of the present invention may contain food-acceptable food supplement additives such as natural carbohydrates and various flavoring agents as additional ingredients.

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

As the flavoring agent, taumartin; Natural flavors such as stevia such as rebaudioside A or glycyrzin and synthetic flavors such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally used in the range of 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 bee nutrients, vitamins, minerals, synthetic flavors and natural flavoring agents, colorants and neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloids Thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the health functional food of the present invention may contain flesh for preparing natural fruit juice, fruit juice beverage, vegetable beverage and the like. These components can be used independently or in combination. The proportion of such additives is generally selected from 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

Hereinafter, the specific method of the present invention will be described in more detail with reference to Examples. The following examples are only one preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

EXAMPLE

Example 1 Preparation of Lyophilized Powders for Bee Breeding, Larvae, Pupa and Adults

Bees make up a collective society, consisting of queen bees, worker bees, and bees. They go through larvae, pupa and adult stages. Therefore, in the present embodiment, the bees larvae, pupa, and adults were recovered from various rods, and then frozen and killed at -20 ° C, and then lyophilized at -50 ° C. Prepared with (FIG. 1). The color difference of the prepared powder is shown in Table 1, and the adult powder showed a low brightness, yellowness and high redness unlike larvae and pupa, and was clearly distinguished from other samples.

[Table 1] Color difference analysis of honeybee larvae, pupa and adult powder

Example 2: Preparation of extracts of bee larvae, pupa, adult powder and component analysis of extracts

Ethanol extract was prepared from the honeybee larvae, pupa and adult powder prepared in Example 1. At this time, 10 times of ethanol was added to each sample, extracted once at room temperature, the extracts were collected and filtered, and concentrated under reduced pressure to prepare a powder. Component analysis of the prepared extracts measured the total polyphenols, flavonoids, total sugars and reducing sugars. The total polyphenol content was added to 400μl of the extracted sample solution, 50μl of Folin-ciocalteau and 100μl of Na ₂ CO ₃ saturated solution, which was allowed to stand at room temperature for 1 hour and then absorbance at 725nm. Tannic acid was used as the standard reagent. For the total flavonoid content, each sample was extracted by stirring for 18 hours with methanol, 400 ml of the filtered extract sample was added 4 ml of 90% diethylene glycol, 40 µl of 1N NaOH was added thereto, and the absorbance was measured at 420 nm after 1 hour of reaction at 37 ° C. Rutin was used as a standard reagent. Reducing sugar was determined by DNS method and total sugar was determined by phenol-sulfuric acid method.

[Table 2] Extraction efficiency and component analysis of honeybee larvae, pupa, adult

As a result, as shown in Table 2, the extraction efficiency of the larva and pupa were similar to 21.8 ~ 22.2%, but the adult was 14.16%, yielding about 60% of the larvae. In the analysis of total polyphenol content of each extract, 12.4 mg / g of honeybee adults were significantly higher than 4.5-5.8 mg / g of larvae and pupa. The total flavonoid content was 4.5 ~ 4.7mg / g in pupa and adult, and 5.8mg / g in larva. However, unlike the total polyphenol content, total flavonoid content was not statistically significant. On the other hand, in the case of total sugar and reducing sugar, the adult extract showed 15-63 times higher and 20-26 times higher contents than larva and pupa extract, respectively. These results indicate that in the case of bees, larvae, chrysalis and adult extracts are significantly different.

Example 3: Evaluation of blood coagulation inhibitory activity of bee larva, pupa, adult extract

The anticoagulant inhibitory activity (thrombogenesis inhibitory activity) of the bee ethanol extract prepared in Example 2 was evaluated, and previously reported methods (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 epitaxial time were measured and evaluated. Plasma was used as a commercial control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and thrombin time, prothrombin time and episode time measurements were performed as follows.

Thrombin Time

50 μl of 0.5 U thrombin (Sigma Co., USA), 50 μl of 20 mM CaCl ₂ , and 10 μl of various concentrations of the sample extract were mixed in a tube of Amelung coagulometer KC-1A (Japan) for 2 minutes, followed by 100 μl of plasma. After addition, the time until plasma coagulates 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 solidification time of 30.5 seconds. The thrombin inhibitory effect was expressed as the average value of the experiment repeated three or more times, and the thrombin inhibitory activity was expressed as the coagulation time when the sample was added divided by the coagulation time of the solvent control.

Prothrombin time

70 μl of standard plasma (MD Pacific Co., China) and 10 μl of various concentrations of sample solution were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 minutes, and then 130 μl of PT reagent was added and the plasma coagulated. The time until 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 solidification time of 16.7 seconds. The prothrombin inhibitory activity was expressed as the coagulation time at the time of sample addition divided by the coagulation time at the solvent control.

aPTT (activated Partial Thromboplastin Time)

100 μl of plasma and 10 μl of various concentrations of the sample extract were added to a tube of Amelung coagulometer KC-1A (Japan), warmed at 37 ° C. for 3 minutes, and then 50 μl of aPTT reagent (Sigma, ALEXIN ^TM ) was added. Incubate for 3 minutes. Thereafter, 50 μl CaCl ₂ (35 mM) was added and the time until the plasma coagulated was measured. DMSO was used as a solvent control instead of the sample, in which case it showed a solidification time of 58.1 seconds. The results of aPTT were shown as the average value of three repeated experiments, and the blood coagulation factor inhibitory activity was expressed as the aPTT at the time of sample addition divided by the aPTT of the solvent control.

[Table 3] Blood Coagulation Inhibitory Activities of Beetle Larvae, Pupa and Adult Ethanol Extracts

As a result, as shown in Table 3, bee larvae, pupa and adult extracts measured thrombin time, prothrombin time, and epitaxial time at a concentration of 5 mg / ml, and showed significant antithrombotic activity in all samples. Did not appear. Commercial antithrombotic aspirin (trade name: Protect), used as a control, extends thrombin time, prothrombin time, and apitime to 1.43-fold, 1.73-fold, and 1.42-fold, respectively, at a concentration of 1.5 mg / ml. Activity was shown.

Example 4: Human platelet aggregation inhibitory activity of bee larva, pupa, adult ethanol extract

Human platelet aggregation inhibitory activity was evaluated for extracts of honey bee samples prepared in Example 2 and the results are shown in Table 4. Platelets are discoid small cells that circulate blood vessels along with various blood cells. They have a cytoplasmic granule that contains high concentrations of various substances related to vascular damage protection and platelet aggregation instead of the nucleus. It is an important cell that secretes factors and combines with collagen exposed to damage of endothelial cells to form a primary hemostatic plug to initiate thrombogenesis. Therefore, platelet aggregation is a very important activity for preventing thrombus formation. to be. 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). After resuspending 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 resuspended at 3,000 rpm for 10 minutes. After centrifugation and resuspended in the suspending buffer, the platelet count was adjusted to 4x10 ⁹ / ml. Then, 2.5 μl collagen was added to the 1 ml suspension for 5 minutes, and platelet aggregation was measured at 37 ° C. using a whole-blood aggregometer (Chrono-log, USA).

[Table 4] Human platelet aggregation inhibitory activity of larvae, pupa and adult ethanol extracts of bees

As a result, as shown in Table 4, in the case of DMSO as a solvent control, platelets were rapidly and strongly aggregated by collagen addition, and aspirin, a platelet aggregation inhibitor, strongly inhibited platelet aggregation in a concentration-dependent manner. In this case, aspirin showed a concentration of 54.3% at a concentration of 0.25mg / ml and a concentration of 29.5% at a concentration of 0.50mg / ml, confirming the basis for use as an antithrombotic agent in the clinic. On the other hand, bee larvae and chrysalis extracts promoted platelet aggregation rapidly and strongly at a concentration of 0.25 mg / ml, resulting in a degree of aggregation of 235-240%. In other words, bee larvae and pupa extract was confirmed that can be used as a hemostatic agent because it promotes blood clot production as opposed to inhibition of blood clot production. However, adult extract had little effect on platelet aggregation at 0.25 mg / ml concentration and showed a similar degree of aggregation to 102.5% of the control.

Example 5 Sequential Organic Solvent Fraction of Adult Bee Ethanol Extract and Component Analysis of Fraction

In the case of bee larvae and pupa extract through the experimental results of Examples 3 and 4, it was confirmed that the development of anti-thrombotic agents due to the thrombus production promoting activity, hexene, ethyl acetate, butanol Sequential organic solvent fractions were collected, and the water residue was finally recovered. The organic solvent fractionation efficiency and the component analysis results of the fractions are shown in Table 5.

Table 5 Preparation of Sequential Organic Solvent Fractions from Bee Ethanol Extracts and Component Analysis of Fractions

As shown in Table 5, 51.4% of the adult honey bee extract was transferred to the hexene fraction, and it was found that most of the adult extract was a lipid component. The water residue was 39.6%, butanol fraction 6.4%, and ethyl acetate fraction. Accounted for 4.4%. Determination of total polyphenol and flavonoid content of the fractions showed the highest 35.0 mg / g and 0.4 mg / g in the butanol fraction, with the highest total and reducing sugars of 157.0 mg / g and 183.2 mg / g in the water residue. Indicated.

Example 6 Evaluation of Blood Coagulation Inhibitory Activity of Fractions of Bee Extracts

Blood coagulation inhibitory activity was measured by the same method as the antithrombotic activity evaluation of Example 3 using the organic solvent fraction of the adult honeybee ethanol extract prepared in Example 5.

[Table 6] Evaluation of Blood Coagulation Inhibitory Activity of Fractions of Adult Bee Extracts

As a result, as shown in Table 6, butanol fraction showed coagulation inhibition due to excellent thrombin, prothrombin and blood coagulation factor inhibition, and thrombin time, prothrombin time, and epitaxial time at 5 mg / ml compared to the non-added households. Were extended by 1.55, 1.39, and 1.51 fold, respectively, showing similar anticoagulant activity to aspirin (1.5 mg / ml). In addition, the concentration of 7 mg / ml butanol fraction was extended by 2.01 times, 1.54 times, and 1.98 times the thrombin time, prothrombin time, and epitaxial time, respectively, compared to the no added group, confirming the concentration-dependent excellent anticoagulant activity. This result is considered to be a significant antithrombotic activity compared to the current commercially available antithrombotic aspirin (trade name, protect). In the future, the butanol fraction of bee ethanol extract may be used as an actual antithrombotic agent, especially a blood coagulation inhibitor. I think it can be developed.

Example 7: Human Platelet Aggregation Inhibitory Activity of Organic Solvent Fractions of Adult Bee Ethanol Extracts

Human platelet aggregation inhibitory activity was evaluated for the organic solvent fractions of the adult honeybee ethanol extract prepared in Example 5, and the results are shown in Table 7 and FIG.

TABLE 7 Human Platelet Aggregation Inhibitory Activity of Fractions of Adult Bee Extracts

As a result, hexene and ethyl acetate fractions showed strong platelet aggregation promoting activity similar to adult ethanol extract at 0.25 mg / ml concentration, and water residue and butanol fraction did not affect platelet aggregation. Therefore, the butanol fraction of the adult ethanol extract exhibited excellent anticoagulant activity and did not affect platelet aggregation.

Example 8: Evaluation of Plasma, Acid and Thermal Stability of Butanol Fraction of Bee Ethanol Extract

The butanol fraction of the adult honeybee ethanol extract obtained in Example 5 was confirmed plasma stability, thermal stability and acid stability against anticoagulant activity. The butanol active fraction showed excellent activity without loss of anticoagulant activity even at 1 hour heat treatment at 100 ° C., 1 hour treatment at pH 2 (0.01 M HCl), and 1 hour treatment in plasma. Therefore, considering the component analysis results of the fraction of Example 5, the characteristics of the organic solvent fraction, and the stability results, the active substance of the butanol fraction of honeybee ethanol extract is expected to be a glycoside of the heat-resistant phenolic compound.

Claims (3)

A pharmaceutical composition for preventing or treating thrombosis, comprising a butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient. Blood coagulation inhibitor containing butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient. Health functional food for the prevention or improvement of thrombosis containing butanol fraction of bee ( Apis mellifera ) ethanol extract as an active ingredient.

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