KR102150122B1 - Antioxidant composition comprising extract of Apis mellifera male pupa - Google Patents

Abstract

The present invention relates to a composition for antioxidants, characterized in that it contains the western species bee ( Apis mellifera ) pupae, and the composition containing the western bee pupae extract is excellent in the inhibitory effect of DPPH to enhance the physiological activity in the body It can be easily used as various pharmaceutical compositions and health functional foods.

Description

[Antioxidant composition comprising extract of Apis mellifera male pupa}

The present invention relates to an antioxidant composition containing a number of pupae of Western bees ( Apis mellifera ).

Today we live in an era of food shortage. In order to solve the world's food problem, genetically modified crops that harm health have occupied the table, and the use of chemical pesticides and chemical fertilizers that harm the environment for large yields causes environmental pollution and causes many side effects to health. In addition, due to energy problems, climate change problems such as drought, floods, heavy rain, and greenhouse gas problems related to climate change, the world's crops are decreasing, and a third of the world's crops are reduced to feed for livestock. Is being consumed. In addition, many livestock generate large amounts of methane gas and carbon dioxide, increasing greenhouse gases, which are the main culprit of global warming, and are facing serious environmental and resource crises. Therefore, there was a need to minimize the consumption of livestock products, save the environment, and develop high-quality alternative food resources. Insects have an average of more than twice the protein content of meat and contain minerals, essential and non-essential amino acids, and vitamins. It is abundant, and since most of the fat is made of water-soluble fat, nutrients can be easily absorbed by the human body, and thus it is gaining popularity as a substitute for protein obtained from livestock products.

In Korea, after the enactment of the Insect Industry Promotion Act in 2010, it is necessary to expand the area of the insect industry.However, until now, many studies have been conducted on pollen mediation, learning, emotions, natural enemies, and environmental purification, but food and feed, which are fields that can greatly expand the industry. , Medicine, biomimetic, and biotechnology research is still insufficient.

Among these insects, earthworms, centipedes, white snails, slugs, wasps, and the like have been mainly used for medicinal purposes since ancient times, and rice grasshoppers, silkworms, pupaes, etc. have been used as foods for a long time. In recent years, larvae and crickets of brown and white-spotted radish have been registered as temporary food ingredients, and among them, brown and crickets have been officially recognized as food ingredients. In order to use other insects as additional food ingredients, various studies are currently being actively conducted.

Accordingly, the inventors of the present invention were studying insects that can be used as various pharmacological ingredients including food, and the pupae of Western bees, which were discarded as unnecessary in beekeepers, are rich in various nutrients and have antioxidant properties, so they can be used as food ingredients or cosmetic ingredients. The present invention was completed by confirming the availability.

Republic of Korea Patent Laid-Open Patent No. 10-2018-0006699 (Name of the invention: Antioxidant, skin whitening or cosmetic composition for improving skin inflammatory diseases containing a brown mealworm extract as an active ingredient, Applicant: Human Cosmetics Co., Ltd., Publication date: January, 2018 19th) Republic of Korea Patent Registration No. 10-0801550 (Name of invention: method of purifying adenosine derivative compounds from silkworm pupae and composition for erectile dysfunction containing adenosine derivative compounds, Applicant: Korea, registration date: January 30, 2008)

It is an object of the present invention to provide a composition for antioxidants containing several pupae of Western species bees ( Apis mellifera ).

The present invention relates to a composition for antioxidants, characterized in that it contains the western species bee ( Apis mellifera ) or an extract thereof.

The extract may be prepared by extracting the western honey bee pupae using water, C1 ~ C4 alcohol, or a mixed solution thereof as a solvent.

The C1 ~ C4 alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and isobutanol.

The C1 ~ C4 alcohol may in particular be ethanol.

In the extraction, the solvent may be 1 to 40 times the volume based on the weight of the pupae.

Accordingly, the present invention provides a health functional food or cosmetic composition for antioxidants, characterized in that it contains a number of pupae or an extract thereof of Western species bee ( Apis mellifera ).

In addition, the present invention comprises the steps of washing and dehydrating the pupae of Western species bees ( Apis mellifera ); Powdering the pupae after drying; And, adding water, C1 ~ C4 alcohol or a mixed solution thereof to the powdered pupae, extracting, and then concentrating and drying the filtrate obtained by removing the ingredients; Preparation of a Western bee pupa extract, characterized in that it comprises Provides a way.

Hereinafter, the present invention will be described in detail.

The extract of the western bee can be extracted using water, C1 ~ C4 alcohol or a mixed solution thereof as a solvent, and the C1 ~ C4 alcohol is methanol, ethanol, propanol, isopropanol, butanol and isobutanol. It may be selected from the group consisting of.

Water, C1 ~ C4 alcohol, or a mixed solution thereof used in the preparation of the extract of the western species bee pupae may be 1 to 40 times the volume (1 to 40ℓ based on 1 kg) based on the weight of the western species bee pupae, and preferably 5-40 times the volume can be used. The extraction conditions of the western species bee pupa extract may be 1 minute to 48 hours at 20 to 100°C. The process can be repeated up to 1 to 4 times.

In addition, as a conventional method in the art, after dissolving water, C1 ~ C4 alcohol or a mixed solution extract thereof of the Western bee pupa in water, selected from the group consisting of n-hexane, chloroform, ethyl acetate and n-butanol. It may be further fractionated using one or more solvents to prepare a fraction.

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

The temperature for preparing the extract or a fraction thereof may be 20 to 100°C, but is not limited thereto. The extraction or fractionation time is not particularly limited, but is preferably extracted within 10 minutes to 2 days, and a conventional extraction device, an ultrasonic grinding extractor, or a fractionator may be used as an extraction device. The thus-prepared Western honey bee pupa extract or fraction may be hot air dried, vacuum dried, or freeze-dried to remove the solvent. In addition, the extract or fraction of the western honey bee can be used after purification using column chromatography.

The Western honey bee pupa extract is a known method used for separating and extracting plant components, such as extraction with an organic solvent (alcohol, ether, acetone, etc.), distribution of hexane and water, and a method by column chromatography according to a commercial method. It can be used alone or after fractionation or purification using a method in combination suitably.

The chromatography was performed on silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, and 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 antioxidant containing the western species bee pupae or extracts thereof. The western species bee pupa or extract thereof may be added in an amount of 0.001 to 100% by weight to the pharmaceutical composition of the present invention.

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

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and 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 one of ordinary skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is from 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection. Since the extract of the present invention has almost no toxicity and side effects, it is a drug that can be safely used even when taken for a long time for prophylactic purposes.

In addition, the present invention provides a health functional food for antioxidant comprising a western species bee pupae or an extract thereof, and food additives acceptable for food. The western species bee pupa or extract thereof may be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes the form of tablets, capsules, pills, liquids, etc., and foods to which the western species bee pupa or extract thereof of the present invention can be added include, for example, various drinks, meat, Sausages, breads, candies, snacks, noodles, ice cream, dairy products, soups, ion drinks, beverages, alcoholic beverages, gum, tea and vitamin complexes.

In addition, the present invention provides an antioxidant cosmetic composition comprising the western species bee pupae or extracts thereof. The formulation of the cosmetic composition is preferably skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, It may be selected from the group consisting of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers. The cosmetic formulation may further include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingo lipids, and seaweed extract, and various excipients included in conventional cosmetics may be further added.

The present invention relates to a composition for antioxidants, characterized in that it contains the western species bee ( Apis mellifera ) pupae, and the composition containing the western bee pupae extract is excellent in the inhibitory effect of DPPH to enhance the physiological activity in the body It can be easily used as various pharmaceutical compositions and health functional foods.

Figure 1 is a graph showing the results of confirming the DPPH inhibitory activity of each of the ethanol extracts of the western species bee ( Apis mellifera ), the male bee larva, the worker bee larva, and the first pupa.

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 species bee pupa>

Western species bee ( Apis mellifera ) 21-24-day-old pupae produced from beehives of a farmhouse were collected and used for component analysis. The pupae were stored in a -20°C freezer immediately after collection.

<Example 2. Analysis of components of western species bee pupa>

Example 2-1. General component analysis

Analysis of the general components of the pupa was conducted according to 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 the atmospheric pressure heating drying method using a 105°C dryer, and the ash content was a direct batch method in which the sample was heated at 550 to 600°C until a white to grayish white ash was obtained. The crude protein content was decomposed by adding a proteolysis accelerator and sulfuric acid to the sample using the Kjeldahl method, and then the content was measured with an automatic protein analyzer of FOSS kjeltec 8400 (Fisher Scientific, Hampton, NH, USA). Crude fat content analysis was performed using the Soxhlet method using petroleum ether as an extraction solvent. Carbohydrate content was expressed as the remaining value after subtracting 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 analysis, but in consideration of changes in the composition of the pupae, the frozen sample was melted without drying, and then homogenized and used for analysis. Table 1 shows the analysis results of ash and moisture, including the three major nutrients of carbohydrate, crude fat, and crude protein present in the pupae. The ash content of the pupae was 0.85% and the moisture content was 74.23%, showing the highest content, and the most important carbohydrates, fats, and proteins for nutrition of organisms were 5.6%, 8.19% and 11.05%, respectively, with the highest protein content. As a result, it was confirmed that the pupaegi were evenly distributed among the three major nutrients required as a food, and especially because of their high protein content, it could be used as a health functional food that helps patients maintain and recover physical strength.

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 structural unit of protein in the body, act as a catalyst for various metabolisms, 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 as the most important measure for evaluating the nutritional value of food proteins (Sakami and Harrington, 1963).

According to the health functional food test method of the Health Functional Food Code, 16 kinds of amino acids such as alanine are decomposed using 6N hydrochloric acid containing _2- mercaptoethanol (C ₂ H ₆ OS), and then hydrochloric acid is decomposed. After removal, sodium citrate buffer was added and filtered, 16 kinds of amino acids were simultaneously analyzed using an automatic amino acid analyzer (Hitachi L-8900, Tokyo, Japan) equipped with an ion exchange column.

Cysteine analysis was performed by adding formic acid to the sample, allowing it to stand for about a day, adding water and lyophilizing it, hydrolyzing it with 6N hydrochloric acid containing 2-mercaptoethanol, and derivatizing it, and performing HPLC with octadecyl silica column Analyzed using. The mobile phase is (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 The conditions were set to a slope condition of ~33 minutes, and the analysis conditions were set at the detection wavelength Ex 262nm, Em 338nm, column temperature 40°C, and flow rate 1.5mL/min.

Tryptophan was hydrolyzed at 135° C. for 22 hours after adding soluble starch and sodium hydroxide solution to the sample, freezing, degassing and sealing. The hydrolyzate was neutralized with hydrochloric acid, centrifuged, and the supernatant was used as an analysis sample, and the content was measured using an automatic amino acid analyzer.

Referring to Table 2, as a result of analyzing the amino acid composition of the male bee pupa, 18 kinds of amino acids were detected, and glutamic acid (Gollnitz and Wiechert, 1967), which activates the function of the brain, showed the highest content of 1631.9mg/100g.

Next, aspartic acid (Sakami and Harrington, 1963), which has functions of excreting ammonia in the body and protecting liver, increases fatigue resistance by participating in nitrogen metabolism and energy metabolism, was identified as 989.4mg/100g, and the remaining 17 amino acids were 17.3. It was measured to be ~731.5mg/100g.

The pupae contain 9 essential amino acids such as valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, threonine, lysine, and histidine, and 731.5mg of leucine (Dohm et al ., 1985), which is used as an energy source for muscles, is used. It showed the highest content in /100g. Therefore, since 19 kinds of amino acids are contained in the pupae, it can be confirmed that the availability as a food having high quality protein is sufficient.

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. Inorganic 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 body's skeleton, control of osmotic pressure, maintenance of muscle function, and energy metabolism (Blumfield et al., 2013).

Among inorganic substances, sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), and phosphorus (P) are Dry and carbonize the sample according to the general test method and dry decomposition method suggested in the Health Functional Food Test Method of the Health Functional Food Code, completely incinerate at 450 to 550°C, add hydrochloric acid, filter through a glass filter, and then use an inductively coupled plasma luminescence analyzer ( ICAP 7400 Duo, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the content. Molybdenum (Mo) and selenium (Se) were also prepared by dry decomposition, and Agilent ICP-MS 7700 (Santa Clara, CA, USA) was used for content analysis. In the analysis of chlorine (Cl), a sample of an amount containing 1 g of salt was incinerated, dissolved in water, filtered, added potassium chromate TS, and titrated with a silver nitrate solution to calculate the amount of chlorine.

As for the inorganic substances present in the pupae, as shown in Table 3, 12 types of inorganic substances were found. Potassium and phosphorus involved in energy metabolism were 235.78mg/100g and 177.73mg/100g, respectively, with 10 different types. It was found to be 4 times higher than that of inorganic matter.

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 the food of Western honeybees, are substances that worker bees collect and store in honeycombs, and are reported to contain amino acids and various inorganic substances (Kim et al., 2017; Hong et al., 2017), and inorganic substances are synthesized in the body. From the point of not, it is understood that minerals are accumulated in the body from honey and pollen consumed before the bee larva becomes an adult and exist 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).

Pretreatment and content analysis of samples were performed according to the vitamin test method of the Health Food Code. For vitamin A, ethanol, pyrogallol ethanol, and potassium hydroxide solution were added to the sample, saponified, and fractionated by adding water and petroleum ether, and then a petroleum ether layer was taken, concentrated under reduced pressure, and isopropanol was added to the sample. Vitamin B ₁ was used as a test solution by adding trichloride acetic acid solution to the sample, homogenizing it, centrifuging to take the supernatant, adding sodium acetate solution and cardiastase solution, and then leaving at 37°C for 10 hours. Vitamin B ₁₂ was used for analysis by adding a phosphate buffer solution to the sample, extracting it by ultrasonication, centrifuging at 21,000 rpm, adding chloroform to the lower layer excluding the precipitate to remove the fat layer, and filtering the remaining water layer through a membrane filter.

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. Vitamins D ₂ and D ₃ were mixed with pyrogallol ethanol to the specimen with shaking, added 90% potassium hydroxide and saponified, and then fractionated with hexane. A hexane layer was taken, concentrated under reduced pressure, methanol was added, filtered through a membrane filter, and used as a test solution.

As for vitamin E, hexane was added to the sample, extracted, filtered, and used for analysis. The content of vitamin E was calculated from the standard calibration curve of α- tocopherol. For quantitative analysis of 7 vitamins, HPLC (Shiseido Nanospace SI-2, Tokyo, Japan) was used, and the analysis conditions such as column, column oven temperature, flow rate, injection volume, and mobile phase were as follows: 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 the pupae were 0.48mg/100g and 0.22mg/100g of water-soluble vitamins B ₁ and C, respectively, 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, and 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 amounts in vegetable palm oil and coconut oil (Verallo-Rowell et al ., 2016). Saturated fatty acids are also known to cause cardiovascular disease when excessive intake (Mozaffarian et al ., 2010).

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

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

Referring to the results of Table 5, the saturated fatty acid present in the pupae is 3.14g/100g, which is not only less than the saturated fatty acid content of pork (pork belly, 15.47%) and beef (4.67%), which are known as major protein sources. It is lower than the content of mackerel (3.96%), a product that is often found on the table of Koreans (Rural Development Administration, 2011), and it contains 2.31g/100g of unsaturated fatty acid, which is 2%, so it can be sufficiently used as a food ingredient. .

<Example 3. Preparation and sensory evaluation of water pupa powder of edible western species>

After washing the pupae of the western bee, the water was removed (dehydrated), and then sterilized at 121°C for 30 minutes and then lyophilized to a moisture content of 8-15%, and prepared as a powder. The taste, aroma, and preference of this powder were compared with the pupa powder of the silkworm, evaluated by 50 people, and shown in Table 6 by the 5-point dot method (5 points: very good-1 point: very bad). As a result, it was confirmed that the silkworm pupa powder had similar preference, taste, and aroma.

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

<Example 4. Confirmation of antioxidant efficacy of western species bee pupa>

Antioxidant activity was measured using vitamin C as a positive control using the DPPH method. After washing and removing water (dehydration) after washing the male bee pupa, the male bee larva, the worker bee larva, and the worker bee pupa of the western species, it was freeze-dried to a moisture content of 8-15% and prepared as a powder. After adding 10 liters of ethanol per 1 kg each for 10 hours to powdered pupae (swine pupa), bee larva, worker bee larva, and worker bee pupa, centrifuge at 8000 rpm for 10 minutes to take only the extract, and concentrate and dry the extract under reduced pressure. As a result, ethanol extracts of the western species bee pupa (beep pupa), bee larvae, worker bee larva, and worker bee pupa were obtained.

DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging ability was measured using the method of Cho et al. (2011). Each ethanol extract was mixed with 0.1 ml of a sample dissolved in ethanol for each concentration and 0.1 ml of a 0.4 mM DPPH solution, and then reacted in the dark for 30 minutes. The absorbance value was measured at 515 nm of a microplate reader (Molecular Devices, Sunnyvale, CA, USA), and the DPPH radical scavenging ability was calculated using the following equation.

DPPH radical scavenging activity (%) = [(Ac-As) / Ac] × 100

Ac: absorbance of the blank sample, As: absorbance of the sample group

As a result, as shown in the result of FIG. 1, it can be seen that the extract of the western breeding bee has remarkably excellent antioxidant activity for inhibiting DPPH compared to the extract of each of the male bee larva, worker bee larva, and worker bee pupa.

<Example 5. Cytotoxicity confirmation>

Cytotoxicity was confirmed using the extract of the western species bee prepared in Example 4. To this end, cytotoxicity to rat red blood cells was confirmed.Rat erythrocytes were washed three times with phosphate-sodium chloride buffer (PBS, pH 7.4), and then phosphate-sodium chloride buffer (PBS, pH 7.4). ) To prepare a 5.0% red blood cell solution. Thereafter, a 5.0% red blood cell solution was dispensed into a 96-well plate by 100 µl, and a solution obtained by diluting each extract in stages was mixed. Subsequently, a phosphate-sodium chloride buffer (PBS, pH 7.4) was added to all the tubes so that the total amount was 200 µl, and incubated in an incubator at 37°C for 30 minutes. After the cultivation was completed, 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 disruption ability for red blood cells was analyzed by measuring the absorbance under a wavelength of 550 nm with an ELISA reader. And as a control, the value when treated with 0.1% (w/v) Triton X-100 was calculated as 100% destructive ability, and when only red blood cells were added, it was calculated as 0% destructive ability. As a result of the experiment, it was confirmed that the extract of the present invention had no ability to destroy red blood cells at a concentration of 1 to 1000 μg/ml (graph not shown).

<Formulation Example 1. Pharmaceutical preparation-preparation of tablets>

200 g of the ethanol extract of the western honey bee pupa 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 a 10% gelatin solution to this mixture, it was pulverized and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.

<Formulation Example 2. Food Manufacture>

Formulation Example 2-1. Preparation of cooking seasoning

The freeze-dried powder of the sterilized western honey bee pupa of the present invention was added to the cooking seasoning in an amount of 1% by weight to prepare a cooking seasoning for health promotion.

Formulation Example 2-2. Manufacture of flour food

Freeze-dried powder of the sterilized western honey bee pupa of the present invention was added to flour in an amount of 0.1% by weight, and bread, cakes, cookies, crackers and noodles were prepared using this mixture to prepare food for health promotion.

Formulation Example 2-3. Preparation of soups and gravies

The freeze-dried powder of the sterilized Western honey bee pupa of the present invention was added to soups and broth in an amount of 0.1% by weight to prepare soups and juices for health promotion.

Formulation Example 2-4. Manufacture of dairy products

The ethanol extract of the western honey bee pupa of the present invention was added in an amount of 0.1% by weight to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

Vegetable juice for health promotion was prepared by adding 0.5 g of ethanol extract of the western species honey bee of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Fruit juice manufacturing

Fruit juice for health promotion was prepared by adding 0.1 g of the ethanol extract of the western species honey bee of the present invention to 1,000 ml of apple juice or grape juice.

<Formulation Example 3. Preparation of cosmetic formulation>

An emulsion composition was prepared by mixing the components of Table 7, and it was confirmed that the composition maintains non-irritation through a skin irritation experiment.

ingredient Content (% by weight) Ethanol Extract of Western Honey Bee Pupa 2.0 Sodium dodecyl sulfate 0.5 glycerin 5.0 Propylene glycol 4.0 Tocopheryl Acetate 3.0 Floating paraffin 5.0 Triethanolamine 0.5 Squalane 3.0 Macadamia Nut Oil 2.5 Polysorbate 60 1.0 Sorbitansquitolate 1.0 Carboxyl vinyl polymer 1.5 Purified water Balance system 100.0

Claims (10)

Western species bee ( Apis mellifera ) cosmetic composition for antioxidant, characterized in that it contains the ethanol extract of pupae.
delete delete Western species bee ( Apis mellifera ) antioxidant composition, characterized in that it contains the ethanol extract of the pupa.
delete delete Western species bee ( Apis mellifera ) antioxidant health functional food composition, characterized in that it contains the ethanol extract of the pupae.
delete delete Washing and dehydrating the pupae of Western bees ( Apis mellifera );
Powdering the pupae after drying; And,
Concentrating and drying the filtrate obtained by adding ethanol to the powdered pupae, extracting and removing ingredients;
A method for producing an antioxidant composition containing ethanol extract of western species bees, characterized in that it comprises a.

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