KR20200048055A - Antithrombosis composition comprising extract of Apis mellifera male pupa - Google Patents

Abstract

The present invention relates to an antithrombotic composition characterized by containing Apis mellifera male pupae. The composition containing an extract of Apis mellifera male pupae has an excellent fibrin decomposition effect, thereby being able to be easily used as a pharmaceutical composition and health functional food which can prevent or treat various vascular diseases such as arteriosclerosis, stroke, etc., related to thrombus.

Description

Antithrombosis composition comprising extract of Apis mellifera male pupa}

The present invention relates to an anti-thrombotic composition containing several pupae of Western honey bee ( Apis mellifera ).

Today we live in an era of food shortages. In order to solve the food problems in the world, genetically modified crops that harm health occupy the table, and use chemical pesticides and chemical fertilizers that harm the environment for large yields to cause environmental pollution and cause many side effects to health. In addition, the world's production crops are decreasing due to energy problems, droughts, floods, heavy rains, climate change problems, and climate change-related greenhouse gas problems. Is being consumed. In addition, many livestock face a serious environmental and resource crisis as they generate a large amount of methane gas and carbon dioxide to increase greenhouse gas, a major cause of global warming. Therefore, there is a need to minimize the consumption of livestock products, save the environment, and develop a high-quality alternative food resource. Insects have an average protein content of 2 times higher than meat, and mineral, essential and non-essential amino acids, vitamins, etc. It is abundant, and most of the fat is made of water-soluble fat, so nutrients can be easily absorbed by the human body.

Although Korea needs to expand the scope of the insect industry after the Enforcement of the Insect Industry Promotion Act in 2010, there have been many studies related to pollen meditation, learning, emotion, natural enemies, and environment purification, but food and feed, which are fields that can greatly expand the industry , Pharmaceuticals, biomimetics, and biotechnology are still insufficient.

Among these insects, earthworms, centipedes, white ginseng, slugs, wasps, and the like have been mainly used for medicinal purposes, and rice locusts, silkworms, and pupae have been used as food for a long time. Recently, caterpillars and crickets of brown and white spotted flowers have been registered as temporary food ingredients, among which brown meal and crickets have been officially recognized as food ingredients. Various other researches are actively underway to use other insects as additional food ingredients.

Accordingly, the present inventors studied food and other insects that can be used as various pharmacological ingredients, while the honeybee bee wasted and wasted in a bee farm. The present invention has been completed by confirming that it is available as a food raw material.

Republic of Korea Patent Publication No. 10-0576972 (Name of invention: composition containing bee larvae or extracts thereof, Applicant: Republic of Korea, Registration Date: April 28, 2006) Republic of Korea Registered Patent No. 10-1634268 (Invention name: composition for treating or preventing arteriosclerosis derived from larvae of hind bee, Applicant: Republic of Korea, Registration date: 2016.06.22) Republic of Korea Patent Registration No. 10-1200404 (Invention name: composition for the treatment or prevention of diseases caused by dysfunction of vasodilatory reactions containing insect glycosaminoglycan, Applicant: Republic of Korea, Registration Date: 2012.11.06)

An object of the present invention is to provide an anti-thrombotic composition containing several pupae of the Western honey bee ( Apis mellifera ).

The present invention relates to a composition for antithrombosis, characterized in that it contains several pupae or extracts of the Western honey bee ( Apis mellifera ).

The extract may be extracted with 2-10% (w / v) acidic aqueous solution of Western honey bee pupae.

The acidic aqueous solution may be selected from the group consisting of acetic acid, hydrochloric acid, nitric acid and sulfuric acid, and may preferably be an aqueous acetic acid solution.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of vascular diseases containing the antithrombotic composition.

The vascular disease may be selected from the group consisting of thrombosis, arteriosclerosis, hypertension, angina, myocardial infarction, ischemic heart disease, heart failure, cerebral infarction, cerebral hemorrhage and stroke, which are vascular diseases caused by the production of thrombus.

In addition, the present invention may be related to a health functional food for preventing or improving vascular disease containing the antithrombotic composition.

Thus, the present invention is a step of washing and dehydrating several pupae of the Western honeybee ( Apis mellifera ); Drying the pupae and pulverizing it; And extracting by adding an acidic aqueous solution to the powdered pupae, and then concentrating and drying the filtrate obtained by removing the dry matter.

Hereinafter, the present invention will be described in detail.

When preparing an acidic aqueous solution extract for the Western honey bee pupae, the acidic solution may use 1 to 40 times the volume based on the weight of the pupae, more preferably 4 to 20 times the volume based on the weight of the pupae used . As such an extraction process, after removing the extract extracted with the acidic solution, the process of adding a new acidic solution to the remaining ingredients and extracting it again may be repeated 2-4 times.

In the present invention, when using an acidic solution used as a solvent for preparing a pupa extract, the extraction conditions are preferably 18 to 24 hours at 2 to 6 ° C.

The most preferred solvent for preparing the pupa extract in the present invention is the most efficient and preferable to use an acidic solution, but in another aspect, as a solvent for preparing the pupa extract of the present invention, water, C1 to C4 alcohol or a mixed solution thereof You can also use However, when extracted with an acidic solution, the difference in effect is more than 10 times higher than that with alcohol, and it is remarkably good.

The C1 ~ C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol. The C1 ~ C4 alcohol may be particularly ethanol. As a solvent used in the preparation of the extract of the Western honey bee pupae, when water, C1 ~ C4 alcohol or a mixed solution thereof is used as a solvent, the volume of the Western honey bee pupae is 1 to 40 times the volume based on weight (1 to 40ℓ per 1kg) ) Can be used, and preferably 5 to 40 times the volume. The extraction conditions of the Western honey bee pupa extract may be from 1 minute to 48 hours at 20 to 100 ° C. The above process can be repeated 1 to 4 times.

In addition, as a conventional method in the art, after dissolving water, C1 ~ C4 alcohol or a mixed solution extract of the Western honeybee pupae in water, it is selected from the group consisting of n-hexane, chloroform, ethyl acetate and n-butanol. It can be prepared as a fraction by further fractionation using one or more solvents.

In another method, after suspending Western honeybee pupae with water, C1 ~ C4 alcohol or a mixed solvent thereof, and then suspending the Western honeybee pupae extract obtained by adding water, preferably, the Western honeybee pupae extract After 1 to 1000 times the weight, more preferably 1 to 500 times, most preferably 1 to 50 times water, and suspended, the suspension consists of n-hexane, chloroform, ethyl acetate and n-butanol. It can be prepared from fractions of Western honeybee pupa obtained by adding a solvent selected from the group. The Western honey bee pupa fraction is preferably a hexane layer obtained by suspending the Western honey bee pupa pupa extract obtained by extracting and concentrating the Western honey bee pupae with water, C1 to C4 alcohol or a mixed solvent thereof, and then mixing it with hexane. The concentrate of the chloroform layer obtained by removing the hexane layer and mixing chloroform with the remaining residue (water layer), or the ethyl acetate layer obtained by removing the chloroform layer and mixing ethyl acetate with the remaining residue (water layer) The concentrate may be a concentrate of a butanol layer obtained by mixing butanol with the residue (water layer) after removing the ethyl acetate layer, or a concentrate of the residue (water layer) remaining after removing the butanol layer. On the other hand, the other fractional conditions are not limited, but after preparing a suspension by adding 1 to 50 times the weight of the weight of the erythropolium pordi extract to the Western honeybee pupa extract, hexane in the same amount as the water, It can be fractionated by adding a solvent selected from the group consisting of chloroform, ethyl acetate, and butanol. Also, after removing the hexane layer, chloroform is added to the remaining residue, chloroform layer is removed, ethyl acetate is added to the remaining residue, and ethyl acetate is removed and butanol is added to the remaining residue. Each solvent (chloroform, ethyl acetate or butanol) can be added sequentially and fractionated.

The preparation temperature of the extract or its fraction may be 20 to 100 ° C, but is not limited thereto. The extraction or fractionation time is not particularly limited, but it is preferable to extract within 10 minutes to 2 days, and an ordinary extraction device, an ultrasonic pulverization extractor, or a fractionator may be used as the extraction device. The thus-prepared Western honey bee pupa extract or fraction can be removed by hot air drying, vacuum drying or freeze drying. In addition, the Western honey bee pupa extract or fraction can be purified by column chromatography and used.

The Western honey bee pupae extract is a known method used for separation and extraction of plant components, such as extraction with an organic solvent (alcohol, ether, acetone, etc.), distribution of hexane and water, and method by column chromatography, according to the commercial method. It can be used by fractionation or purification using a single or appropriately combined method.

The chromatography is silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, medium pressure liquid chromatography chromatography, thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

In addition, the present invention provides a pharmaceutical composition for antithrombosis, which contains Western honeybee pupae or extracts thereof. The Western honey bee pupae or its extract may be added to the pharmaceutical composition of the present invention as 0.001 to 100% by weight.

The pharmaceutical composition may be formulated and used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the extract of the present invention, for example, starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of those skilled in the art, and dosages generally range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferred dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be administered once a day, or may be divided into several times. 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 various mammals, such as rats, livestock, and humans. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracranial injection. Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even for a long period of time for prevention purposes.

In addition, the present invention provides a health functional food for anti-thrombosis, including Western honeybee pupae or an extract thereof and a food-acceptable food supplement additive. The Western honey bee pupae or its extract can be added to the health functional food of the present invention at 0.001 to 100% by weight. The health functional food of the present invention includes tablets, capsules, pills or liquids, etc., and foods to which the Western honeybee pupae or extracts of the present invention can be added include, for example, various drinks, meat, Sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, beverages, alcoholic beverages, chewing gum, tea and vitamin complexes.

The present invention relates to an anti-thrombotic composition characterized by containing several pupae of the Western honey bee ( Apis mellifera ), wherein the composition containing the Western honey bee pupae extract has a very good fibrin decomposition effect and arteriosclerosis related to thrombus, It can be easily used as a pharmaceutical composition and health functional food that can prevent or treat various vascular diseases such as stroke.

1 is a result of confirming the thrombolytic activity of the 5% (w / v) acetic acid aqueous solution extract of several pupae of the Western honey bee ( Apis mellifera ).

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

<Example 1. Collection of Western honeybee pupae>

Western honey bee ( Apis mellifera ) The 21-24-day-old pupa produced from the beehive of a speculative farm was collected and used for ingredient analysis. Several pupae were immediately stored in a freezer at -20 ℃.

<Example 2. Component analysis of Western honeybee pupae>

Example 2-1. General component analysis

The analysis of the general composition of several pupae was conducted in accordance with the Food Ingredient Test Method of the Food Code of the Ministry of Food and Drug Safety. That is, the moisture content was measured by a normal pressure heating and drying method using a 105 ° C dryer, and the ash content was a direct batch method in which samples were heated at 550 to 600 ° C until white to grayish white ash was obtained. The crude protein content was decomposed by adding a proteolytic accelerator and sulfuric acid to the sample using the Kjeldahl method, and then the content was measured by an FOSS kjeltec 8400 (Fisher Scientific, Hampton, NH, USA) protein automatic analyzer. In the analysis of crude fat content, Soxhlet method was used as an extraction solvent for petroleum ether. The carbohydrate content was expressed as the remaining value minus crude protein, crude fat, moisture and ash content in 100 g of the sample.

On the other hand, the frozen sample in Example 1 was used for the analysis, but it was homogenized after melting and freezing the frozen sample without drying considering the change in the composition of several pupae. Table 1 shows the analysis results for ash and moisture, including three major nutrients of carbohydrate, crude fat, and crude protein present in several pupae. The ash content of male pupae was 0.85% and the moisture content was 74.23%, which was the most important. Carbohydrates, fats, and proteins, which are the most important for the nutrition of organisms, were 5.6%, 8.19%, and 11.05%, respectively, with the highest protein content. As a result, it was confirmed that several pupae are distributed evenly among the three major nutrients required as food, and especially, the protein content is high, so it can be used as a health functional food that helps patients maintain and recover their fitness.

Component Content (%) Moisture 74.23 ± 1.21 Carbonate  5.68 ± 0.11 Ash  0.85 ± 0.01 Crude protein 11.05 ± 0.08 Crude fat  8.19 ± 0.05

Example 2-2. Amino acid composition and content analysis

Amino acids are the basic building blocks of proteins in the body and act as catalysts for various metabolism and are involved in the regeneration and recovery of human tissues (Sakami and Harrington, 1963). Essential amino acids are amino acids that are not synthesized in the body and are known to be the most important measure of the nutritional value of food proteins (Sakami and Harrington, 1963).

According to the health functional food test method before the health functional food, 16 kinds of amino acids such as alanine were decomposed protein in the sample using 6 N hydrochloric acid containing _2- mercaptoethanol (C ₂ H ₆ OS), and then hydrochloric acid was used. After removal and filtering by adding sodium citrate buffer, 16 amino acids were simultaneously analyzed using an amino acid analyzer equipped with an ion exchange column (Hitachi L-8900, Tokyo, Japan).

For cysteine analysis, formic acid was added to the sample, and after standing for about a day, water was added and lyophilized, followed by hydrolysis using 6N hydrochloric acid containing 2-mercaptoethanol and derivatization to obtain HPLC with octadecyl silica column. Analysis. Mobile phase (A) 40 mM NaH ₂ PO ₄ and (B) MeCN: MeOH: H ₂ O = 45: 45: 10 solution (A) 95-44%; 0-31 min, (A) 44%; 31 It was set to the gradient condition of ∼33 minutes, and the analysis conditions were set to detection wavelength Ex 262nm, Em 338nm, column temperature 40 ℃, flow rate 1.5mL / min.

Tryptophan was added to a sample of soluble starch and sodium hydroxide solution, frozen, degassed, sealed and hydrolyzed at 135 ° C for 22 hours. The hydrolyzate was neutralized with hydrochloric acid and centrifuged to use the supernatant as an analysis sample and the content was measured using an automatic amino acid analyzer.

Referring to Table 2, as a result of the amino acid composition analysis of the Western honeybee pupae, 18 amino acids were detected and the highest content was 1631.9mg / 100g for glutamic acid (Gollnitz and Wiechert, 1967), which activates brain function.

Next, 989.4 mg / 100g of aspartic acid (Sakami and Harrington, 1963), which has a function of ammonia discharge and liver protection in the body and increases fatigue resistance by being involved in nitrogen metabolism and energy metabolism, was found to be 17.3. It was measured as ~ 731.5mg / 100g.

The pupae contains nine essential amino acids such as valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, threonine, lysine, and histidine, and 731.5 mg of leucine (Dohm et al ., 1985) used as a muscle energy source. / 100g showed the most content. Therefore, it can be confirmed that the availability of foods having high-quality protein is sufficient because 19 kinds of amino acids are contained in several pupae.

        Amino acid Content (mg / 100g)         Alanine  501.7 ± 3.1 Valine ^*  496.8 ± 1.8 Leucine ^*  731.5 ± 4.1 Isoleucine ^*  423.4 ± 1.1         Proline  524.5 ± 2.2 Phenylalanine ^*  389.8 ± 2.1 Trytophan ^*   77.0 ± 0.6 Methionine ^*   24.7 ± 0.2         Glysine  441.7 ± 1.8         Serine  439.2 ± 3.1 Threonine ^*  425.7 ± 1.5         Cystine   17.3 ± 0.2         Tyrosine  392.6 ± 2.3         Aspartic acid  989.4 ± 4.4         Glutamic acid 1631.9 ± 6.2 Lysine ^*  665.2 ± 1.5         Arginine  486.3 ± 1.2 Hystidine ^*  248.6 ± 1.0 ^* Essential amino acid

Example 2-3. Mineral composition and content analysis

Minerals are substances that are not synthesized in the body, such as essential amino acids, and are classified as micronutrients necessary for the maintenance of biological functions, and are known to have various functions such as the formation of the skeleton of the body, regulation of osmotic pressure, maintenance of muscle functions, and energy metabolism (Blumfield et al., 2013).

Among the minerals, sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), and phosphorus (P) After drying and carbonizing the sample according to the dry decomposition method presented in the General Test Method and the Health Functional Food Test Method before the Health Functional Food Test, and completely painting at 450 ~ 550 ℃, add hydrochloric acid, filter with a glass filter, and then conduct an inductively coupled plasma luminescence analyzer ( ICAP 7400 Duo, Thermo Fisher Scientific, Waltham, MA, USA). Molybdenum (Mo) and selenium (Se) were also pre-treated by dry decomposition, and the content analysis was performed using Agilent ICP-MS 7700 (Santa Clara, CA, USA). In the analysis of chlorine (Cl), a sample containing an amount of 1 g of salt was painted, dissolved in water, filtered and potassium chromate solution was added, followed by titration with silver nitrate solution to calculate the amount of chlorine.

As shown in Table 3, the minerals present in several pupae were found to have 12 kinds of minerals. Potassium and phosphorus involved in energy metabolism were 235.78mg / 100g and 177.73mg / 100g, respectively. It was found to be four times higher than the mineral.

Mineral Content (mg / 100g) Na  25.99 ± 0.18 K 235.78 ± 1.12 Cl  39.50 ± 0.21 Ca  10.66 ± 0.11 Mg  18.37 ± 0.13 P 177.73 ± 2.44 Fe   1.59 ± 0.02 Zn   1.96 ± 0.02 Cu   0.33 ± 0.01 Mn   0.10 ± 0.01 Mo   0.06 ± 0.01 Se   0.04 ± 0.01

Honey and pollen, which are food for Western-style bees, have been reported to contain amino acids and various minerals as a material collected by honeybees from wheat plants and stored in honeycombs (Kim et al., 2017; Hong et al., 2017). It is understood that minerals are accumulated in the body from honey and pollen consumed until the bee larvae become adults, and are present in several pupae.

Example 2-4. Vitamin composition and content analysis

Vitamins are substances that are involved in the regulation of physiological functions and metabolism in the body, and are essential nutrients that exhibit physiological activity even in adaptive amounts and are classified as fat-soluble and water-soluble vitamins (Khadim, 1981).

Pre-treatment and content analysis of samples were conducted according to the vitamin test method of the Health Food Code. Vitamin A was saponified by adding a solution of ethanol, pyrogallol ethanol, and potassium hydroxide to the sample, fractionated by adding water and petroleum ether, and then the petroleum ether layer was concentrated under reduced pressure and isopropanol was used as an analysis sample. Vitamin B ₁ was used as a test solution by adding triacetic acid solution to the sample, homogenizing it, centrifuging it, taking the supernatant, adding sodium acetate solution and cardiatase solution, and then leaving it at 37 ° C for 10 hours. Vitamin B ₁₂ was added to the sample with a phosphate buffer solution, extracted with ultrasound, and centrifuged at 21,000 rpm to remove the fat layer by adding chloroform to the lower layer excluding the precipitate, and filter the remaining water layer with a membrane filter for analysis.

Vitamin C was homogenized by adding 10% methane phosphate solution to the sample, centrifuged at 3000 rpm, and the supernatant was used as a test solution. Vitamin D ₂ and D ₃ were added to pyrogallol ethanol to the sample, shake-mixed, saponified with 90% potassium hydroxide, and fractionated with hexane. The hexane layer was taken, concentrated under reduced pressure, and then methanol was added and filtered through a membrane filter to be used as a test solution.

Vitamin E was extracted with hexane in the sample, filtered, and used for analysis. The content of vitamin E was calculated from the standard calibration curve of α -tocopherol. Quantitative analysis of 7 kinds of vitamins was carried out using HPLC (Shiseido Nanospace SI-2, Tokyo, Japan) .The analysis conditions such as column, column oven temperature, flow rate, injection amount, mobile phase, etc. It was the same.

Vitamin Content (mg / 100g) A ND ^* B ₁ 0.48 ± 0.01 B ₁₂ ND C 0.22 ± 0.01 D ₂ ND D ₃ ND E 0.10 ± 0.01 K ₁ ND ^* ND: Not detected

Referring to Table 4, the vitamins present in several pupae were found to be 0.48mg / 100g and 0.22mg / 100g, respectively, of water-soluble vitamins B ₁ and C, and fat-soluble vitamin E was found to be 0.10mg / 100g, but vitamins A and B ₁₂ , D ₂ , D ₃ and K ₁ were not detected.

Example 2-5. Fatty acid content analysis

Fatty acid is a hydrolyzate of fat, and refers to a compound having a carboxyl group in the hydrocarbon chain. It is divided into saturated fatty acids and unsaturated fatty acids depending on the presence or absence of a carbon-carbon double bond in the hydrocarbon chain. Saturated fatty acids are biosynthesized in the body and are known to be mainly contained in animal fats, but are also present in large quantities in vegetable palm oil or coconut oil (Verallo-Rowell et al ., 2016). Saturated fatty acids are also known to cause cardiovascular disease when consumed excessively (Mozaffarian et al ., 2010).

The content of saturated fatty acids and unsaturated fatty acids present in several pupae of Western honeybees was carried out according to the fatty acid test method of food industry. That is, the sample was homogenized, the fat extracted with chloroform and diethyl ether was dissolved, and then concentrated with nitrogen. Trifluoroboranmethanol and toluene were added to the concentrate, heated in an oven at 100 ° C., and then cooled at room temperature. Subsequently, water and hexane were added and the separated supernatant was used as a test solution and detected with a capillary column (Sigma-Aldrich SP-2560, St.Louis, MO, USA) in a GC (Agilent 7890A) equipped with a flame ionization detector. Quantitative analysis was performed under the analysis conditions of temperature 285 ° C, flow rate 0.75 mL / min, injection volume 1 μL, oven temperature 100 ° C (4 min), 208 ° C (3 ° C / min), and 244 ° C (15 min).

Type Content (g / 100g)  Saturated fatty acid 3.14 ± 0.03 Unsaturated fatty acid 2.31 ± 0.01

Referring to the results in Table 5, the saturated fatty acid present in several pupae was 3.14 g / 100 g (Table 5), and the saturated fatty acid content of pork (15.47% based on pork belly, 15.47%) and beef (4.67%) known as the main protein sources. It is not only less, but also lower than the content of mackerel (3.96%), a product that frequently rises on Koreans' table (Rural Development Administration, 2011), and contains 2% of unsaturated fatty acid at 2.31g / 100g (Table 5). It seems to be able to use enough.

<Example 3. Preparation of edible western honeybee powder and sensory evaluation>

After washing several pupae of the Western honey bee and removing (dehydrating) it, it was sterilized at 121 ° C for 30 minutes and lyophilized to a water content of 8-15%, and then prepared as a powder. About this powder, the taste, aroma, preference, etc. were compared to silkworm pupae powder and evaluated to 50 persons, and the result was shown in Table 6 by a 5-point scoring method (5 points: very good-1 point: very bad). As a result, it was confirmed that the silkworm pupa powder had similar taste, taste, and aroma.

Condition flavor incense Preference Western honey bee powder 3.2 3.2 3.5 Silkworm pupa powder 3.4 3.6 3.2

<Example 4. Confirmation of antithrombotic efficacy of Western honey bee pupa extract>

10 ml of 5% (w / v) acetic acid aqueous solution was added to 1 g of the pupa powder of the Western honey bee obtained by lyophilization in Example 3 and mixed well, followed by extraction at 4 ° C. for 18 hours with gentle stirring. Filtration was performed using a 0.45 μM fillter to obtain an extract. After dispensing 700 ~ 800µl of the extract in 1.5ml tube by using a vacuum vaccum evaporator (CVC-3100) for 24 hours, finally, several extracts of Western honeybee were obtained. The extract was dissolved again in 0.01% Acetic acid to prepare each extract in a concentration suitable for use in the experiment.

Next, for the thrombolytic activity assay, 8 mm disks were sterilized and prepared in a dried state, followed by mixing fibrinogen and thrombin in 6 wells to form a fibrin state, followed by 2 ml of each well in the fibrin state before hardening. I was busy.

Thereafter, a sterilized disc was placed in the center of the well using tweezers, and 0.12 mg / ml extract was placed on the disc, and 20 μl was added and reacted in an incubator at 37 ° C. and then the process of dissolving the fibrin composition was photographed. , The size of the resulting ring was measured at 24 hours.

A photograph of the result for this is shown in FIG. 1, and the result of FIG. 1 is shown in Table 7. Tables 1 and # 2 of Table 7 show the results for the upper and lower treatment groups in FIG. 1, respectively.

Experimental treatment tool Diameter of the ring in which the thrombus material is dissolved (㎜) Negative control Positive control
(20unit / ml human plasmin, Sigma) Pupa pupa extract
(0.12 mg / ml) # One - 12.96 20.67 # 2 - 12.90 20.99 Average - 12.93 20.83

** Marked with '-' in Table 7 above means no antithrombotic activity.

Referring to FIG. 1 and Table 7, 0.12 mg / mL of pupal pupa extract shows a higher antithrombotic effect than 20 unit / ml of human plasmin (Sigma) used as a positive control (negative control-used for dilution of pupa pupae extract) 5% acetic acid, no antithrombotic effect), and excellent antithrombotic efficacy.

<Example 5. Confirmation of cytotoxicity>

Cytotoxicity was confirmed by using the pupa extract of the Western honey bee prepared in Example 4. To this end, cytotoxicity to rat red blood cells was confirmed. After washing red blood cells (Rat erythrocytes) three times with phosphate-sodium chloride buffer (PBS, pH 7.4), phosphate-sodium chloride buffer (PBS, pH 7.4) ) To prepare a 5.0% red blood cell solution. Then, 100% of the 5.0% red blood cell solution was dispensed into a 96-well plate, followed by mixing the diluted solution of each extract step by step. Subsequently, phosphate-sodium chloride buffer (PBS, pH 7.4) was added so that the total amount of the total solution was 200 μl in all tubes, and the cells were incubated for 30 minutes in a 37 ° C. incubator. After the incubation, the supernatant was transferred to a 96-well plate by centrifugation for 10 minutes at a rotation speed of 1000 rpm in a centrifuge. The destruction ability for red blood cells was analyzed by measuring the absorbance under a wavelength of 550 nm with an ELISA reader. In addition, the value when treated with 0.1% (w / v) Triton X-100 as a control was calculated as 100% destructive ability, and the case where only red blood cells were added was calculated as 0% destructive ability. As a result of the experiment, it was confirmed that the extract of the present invention had no destruction ability of red blood cells at a concentration of 1 to 1000 µg / ml (not shown in the graph).

<Example 6. Comparison of antithrombotic effects according to sample conditions>

The antithrombotic efficacy was confirmed under the same conditions as in Example 5, but extracts were prepared for each period of bees and other felling insects as shown in Table 8, and then compared and shown in Table 8. At this time, the preparation method of the extract was carried out in the same manner as the extract preparation conditions of Example 4. As a result, it was confirmed that the anti-thrombotic efficacy of the extract of Western honey bee pupae of the present invention was the best.

0.12mg / ml of each extract Diameter of the ring in which the thrombus material is dissolved (㎜) Extract of honey bee larvae 12.35 Bumblebee pupa extract from Western honey bees 23.01 Extract of honeybee bee venom adult - Caterpillar extract of Western bee worker bee 10.13 Pupa Extract from Western Bees 11.20 Adult bee extract from Western bees - Caterpillar extract of Western bee queen bee 9.68 Western bee queen bee pupa extract - Adult bee extract of Western bee queen bee - Larva extract of bumblebee bee 12.55 Pupa bee worker bee pupa extract - Adult beetle extract of bumblebee bee 10.05 Larva extract of bumblebee bee 10.95 Pupa bee pupa extract - Adult beetle extract of bumblebee bee - Larva extract of pumpkin bee queen bee 10.63 Pumpkin Bee Queen Bee's Pupa Extract - Pumpkin Bee Queen Bee Adult Extract 12.57 Caterpillar extract of the Western Bee Bee - Pupa Extract of Western Beaver Bee - Adult extracts of the Western Bee Bee 10.12 Larva extract - Pupa pupa extract - Adult extracts of the western back bee 9.86 Caterpillar extract of queen bee queen - Western bee queen bee pupa extract 9.59 Adult extract of Queen Western Bees -

** Marked with '-' in Table 8 above means that there is no antithrombotic activity.

<Formulation Example 1. Pharmaceutical preparation-Preparation of tablet>

200 g of ethanol extract of Western honeybee pupae of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. After adding 10% gelatin solution to this mixture, it was ground and passed through a 14 mesh sieve. This mixture was dried, and a mixture obtained by adding potato starch 160g, talc 50g, and magnesium stearate 5g was purified.

<Formulation Example 2. Food manufacturing>

Formulation Example 2-1. Cooking seasoning

The freeze-dried powder of sterilized Western honey bee pupae of the present invention was added to the cooking seasoning in 1% by weight to prepare a cooking seasoning for promoting health.

Formulation Example 2-2. Production of flour food

The lyophilized powder of sterilized Western honey bee pupae of the present invention was added to wheat flour at 0.1% by weight, and bread, cake, cookies, crackers and noodles were prepared using this mixture to prepare health foods.

Formulation Example 2-3. Preparation of soup and gravy

The lyophilized powder of sterilized Western honey bee pupae of the present invention was added to soups and gravy at 0.1% by weight to prepare health soup and gravy.

Formulation Example 2-4. Manufacturing dairy products

Ethanol extract of Western honeybee pupae of the present invention was added to milk at 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice production

0.5 g of ethanol extract of Western honeybee pupae of the present invention was added to tomato juice or carrot juice to prepare a vegetable juice for health promotion.

Formulation Example 2-6. Fruit juice manufacturing

0.1 g of ethanol extract of Western honeybee pupae of the present invention was added to 1,000 ml of apple juice or grape juice to prepare a fruit juice for health promotion.

Claims (8)

Anti-thrombotic composition, characterized in that it contains several pupae or extracts of Western honey bee ( Apis mellifera ). According to claim 1,
The extract is a composition for anti-thrombosis, characterized in that it extracts 2-10% (w / v) acidic aqueous solution of Western honeybee pupae as a solvent. According to claim 2,
The acidic aqueous solution is an antithrombotic composition, characterized in that it is an aqueous solution of an acid selected from the group consisting of acetic acid, hydrochloric acid, nitric acid and sulfuric acid. A pharmaceutical composition for the prevention or treatment of vascular diseases containing the composition of any one of claims 1 to 3. The method of claim 4,
The vascular disease is a disease selected from the group consisting of thrombosis, arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, cerebral infarction, cerebral hemorrhage and stroke. A health functional food for the prevention or improvement of vascular diseases containing the composition of any one of claims 1 to 3. The method of claim 6,
The vascular disease is a disease selected from the group consisting of thrombosis, arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, cerebral infarction, cerebral hemorrhage and stroke. Washing and dehydrating several pupae of Western honey bee ( Apis mellifera ); Drying the pupae and pulverizing it; And extracting by adding an acidic aqueous solution to the powdered pupae and then concentrating and drying the filtrate obtained by removing the dry matter; a method for preparing an antithrombotic composition comprising the Western honeybee pupae pupa extract.

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