KR20240121357A - Antioxidant and anti-inflammatory composition containing bluegill extract - Google Patents

Abstract

The present invention relates to an antioxidant and anti-inflammatory composition comprising a bluegill extract and a method for producing a bluegill extract. The bluegill extract exhibits excellent antioxidant effects through DPPH radical scavenging activity and ABTS scavenging activity, and has excellent anti-inflammatory effects by inhibiting the production of NO by LPS. Therefore, the composition of the present invention comprising a bluegill extract can be usefully used as an antioxidant and anti-inflammatory composition.

Description

{Antioxidant and anti-inflammatory composition containing bluegill extract}

The present invention relates to an antioxidant and anti-inflammatory composition comprising a bluegill extract and a method for producing a bluegill extract. The bluegill extract exhibits excellent antioxidant effects through DPPH radical scavenging activity and ABTS scavenging activity, and has excellent anti-inflammatory effects by inhibiting the production of NO by LPS. Therefore, the composition of the present invention comprising a bluegill extract can be usefully used as an antioxidant and anti-inflammatory composition.

Bluegill ( Lepomis macrochirus ) belongs to the Centrarchidae family of the Perciformes order. It is native to southeastern North America (Virginia, Florida, Texas, Mexico, New York) and has since been introduced and established throughout North America, Europe, Asia, and South Africa.

The bluegill has a laterally compressed head and body, a tall body height, and a short body length. The number of soft rays in the dorsal fin is 10–12, the number of soft rays in the anal fin is 10–12, the number of lateral line scales is 38–54, and the body shape is oval. The head is relatively large, the eyes are located on the back of the head, the snout is pointed, and the mandible is slightly forward of the maxilla. The margin of the anterior fin has a serrated protrusion. The center of the posterior margin of the caudal fin is slightly concave. The lateral line is complete and parallel to the dorsal outline. The upper half of the body is dark blue, and the ventral side has a yellow sheen. There are 8–9 long brown transverse stripes on the sides of the body. As it grows, the body color changes from dark gray-brown to dark brown, and the transverse stripes become increasingly indistinct. Both males and females have dark blue spots on the slightly protruding part of the back of the gill cover, hence the name bluegill. During the spawning season, males have yellow and orange mating colors with light blue bands. Bluegills are highly reproductive and adaptable, and are used for fishing and food. Because bluegills are highly reproductive and easy to raise, they are also used as standard laboratory animals for fish.

Meanwhile, it has been reported that bluegill directly and indirectly affects the food chain structure of the ecosystem, energy flow of the ecosystem, and habitat changes, thereby causing ecosystem disruption. In Korea, the Fisheries Agency introduced 510 bluegill from Japan in 1969 for experimental aquaculture and released them near Paldang Dam in the Han River. After more than 10 years, they have recently emerged as a dominant species in dam reservoirs such as Paldang Dam, Daecheong Dam, and Andong Dam. As a result, they are causing significant changes in fish diversity by consuming large quantities of native fish species, including fry and shrimp, and countermeasures are being demanded. Due to the negative perception of bluegill as an ecosystem-disrupting species, research on its utilization is low compared to its high nutritional value. In addition, bluegill has a strong fishy smell compared to other fish species, making it difficult to utilize as food or food products.

Accordingly, the inventors of the present invention screened the activity of bluegill extracts to increase the utilization of bluegill, which is classified as an ecosystem-disrupting fish species, and completed the present invention by elucidating that bluegill extracts have excellent antioxidant and anti-inflammatory effects.

Accordingly, the purpose of the present invention is to provide an antioxidant or anti-inflammatory composition containing a bluegill extract.

Another object of the present invention is to provide a method for producing a bluegill extract.

Another object of the present invention is to provide antioxidant or anti-inflammatory uses of bluegill extract.

The present invention relates to an antioxidant or anti-inflammatory composition comprising a bluegill extract, and a method for producing a bluegill extract.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention relates to an antioxidant or anti-inflammatory composition comprising a bluegill extract.

In the present invention, the bluegill extract may be an extract including bluegill oil obtained by low-temperature pressing and extracting bluegill at 55 to 65°C.

In the present invention, the bluegill extract includes not only those obtained by low-temperature pressing extraction, but also those obtained by additionally applying a purification process thereto. For example, the extract includes fractions obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, fractions obtained by various additional purification methods such as separation by various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity), etc.

In the present invention, the bluegill extract can be prepared in a concentrate and/or powder state by additional processes such as reduced pressure distillation and freeze drying or spray drying.

In the present invention, the bluegill extract may be an extract containing bluegill protein obtained through hot water extraction and enzyme treatment.

In the present invention, hot water extraction may be performed at a temperature of 50 to 80°C, 50 to 70°C, 60 to 80°C, or 60 to 80°C.

In the present invention, the enzyme may be alcalase.

In the present invention, the bluegill extract may contain 70 to 90% by volume, 70 to 80% by volume, or 80 to 90% by volume of high-purity unsaturated fatty acids.

The composition of the present invention exhibits an antioxidant effect by scavenging DPPH radicals and ABTS radicals, and therefore can be used as an antioxidant composition.

As we age, aging is accelerated by various factors including active oxygen species, and this is manifested as wrinkles, a representative phenotype of aging. In order to prevent aging, that is, wrinkles, reducing exposure to UV rays, which account for 80% of the cause of aging, and providing sufficient antioxidants are important preventive and therapeutic methods.

Antioxidants are known to prevent aging by reducing oxidative stress in cells by removing reactive oxygen species, and each antioxidant has a different function in influencing aging mechanisms or aging-related mechanisms such as collagen synthesis, thereby suppressing aging and its phenotype, wrinkles. It is also known that antioxidants affect redox signaling in cells, thereby increasing repair activity and preventing skin aging by inducing the activation of repair proteins.

Therefore, the composition of the present invention exhibiting excellent antioxidant activity can be used for wrinkle improvement purposes.

The composition of the present invention has an excellent anti-inflammatory effect by inhibiting the production of nitric oxide (NO) by LPS (Lipopolysaccharide), and therefore can be used as an anti-inflammatory composition.

NO is a cell signaling substance that plays a key role in inflammatory responses, performing various physiological functions such as platelet aggregation and neutrophil assembly in the vascular system and metabolic regulation in skeletal muscle, and is synthesized by many immune-related cells such as macrophages, leukocytes, and T lymphocytes. In the inflammatory process, NO is involved in infection control, regulation of signaling factors and transcription factors, regulation of vascular responses, leukocyte migration, cytokine production, and apoptosis.

In the present invention, the composition may be a cosmetic composition.

In the present invention, the cosmetic composition may have any one formulation selected from among skin, skin softener, skin toner, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, essence, nutrition essence, pack, cleansing foam, cleansing water, cleansing cream, body lotion, body cleanser, soap, and powder, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for formulating the formulation, and the types and amounts of these ingredients can be easily selected by those skilled in the art.

In the present invention, when the formulation of the cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane-butane or dimethyl ether may be additionally included.

In the present invention, when the formulation of the cosmetic composition is a solution or an emulsion, a solvent, a solvating agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

In the present invention, when the formulation of the cosmetic composition is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as carrier components.

In the present invention, when the formulation of the cosmetic composition is a surfactant-containing cleansing, the carrier component may include fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolenic derivative, or ethoxylated glycerol fatty acid ester.

In the present invention, the cosmetic composition may additionally contain excipients including fluorescent substances, fungicides, hydrotropism inducers, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and the like.

In the present invention, the composition may be a food composition.

In the present invention, the food may be a beverage, a gum, a tea, a vitamin complex, a health supplement, and more specifically, may be any one selected from the group consisting of dairy products, confectionery, seasonings, beverages and drinks, snacks, candies, jellies, ice cream and frozen desserts, breakfast cereals, nutrition bars, snack bar chocolate products, processed foods, cereal products and pasta, soups, sauces and dressings, confectionery products, oils and fat products, dairy drinks and milk drinks, tea, soy milk and soy dairy-like products, frozen foods, prepared foods and food substitutes, meat products, cheese, yogurt, bread and rolls, cakes, cookies and crackers.

In the present invention, the food composition can be manufactured and processed in the form of tablets, capsules, powder, granules, liquid, pills, etc.

The content of the bluegill extract as an effective ingredient contained in the food composition of the present invention is not particularly limited, and may be appropriately added in an amount of 0.01 to 15 wt% of the total food weight, and in the case of a health beverage composition, may be added in an amount of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

In the present invention, the beverage composition has no particular limitations on the liquid component other than containing the extract as an essential component in the indicated proportion, and may contain various flavoring agents or natural carbohydrates as additional components like a conventional beverage.

In the present invention, natural carbohydrates are common 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.

In the present invention, the food composition may be a "health functional food composition", and the health functional food composition refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body, and is meant to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.

In the present invention, the health functional food may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and criteria for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. The items listed in the above 'Food Additives Codex' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sucrose pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in the form of a tablet may be made by mixing the effective ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, and then compressing and molding the mixture, or directly compressing and molding the mixture. In addition, the health functional food in the form of a tablet may contain a coupling agent, etc., if necessary. Among the health functional food in the form of a capsule, a hard capsule can be manufactured by filling a mixture of the effective ingredient of the present invention with additives such as excipients into a conventional hard capsule, and a soft capsule can be manufactured by filling a mixture of the effective ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The soft capsule can contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, etc., if necessary. A health functional food in the form of a ring can be prepared by molding a mixture of the effective ingredient of the present invention with an excipient, a binder, a disintegrant, etc., by a conventionally known method, and if necessary, can be coated with white sugar or another coating agent, or the surface can be coated with a material such as starch or talc. Granular health functional foods can be manufactured into granules by mixing excipients, binders, disintegrants, etc. of the effective ingredient of the present invention with a known method, and may contain flavoring agents, flavoring agents, etc., as needed.

In the present invention, the composition may be an animal feed composition.

In the present invention, the animal feed composition may have the effect of enhancing the immune activity of the animal by including a bluegill extract.

In the present invention, the feed composition may contain various feed ingredients known in the art in addition to the bluegill extract as an effective ingredient.

In the present invention, there is no particular limitation on the animal, and for example, it may be a dog or a cat. In particular, when the animal feed composition of the present invention is consumed, in the case of dogs, it may help with skeletal development and growth and development due to the nutrients abundantly contained in the bluegill extract, and in the case of cats, it may help with consuming taurine, an essential amino acid, since cats cannot produce taurine on their own.

Another aspect of the present invention relates to a method for producing a bluegill extract, comprising the following steps:

(a) a sterilization step for sterilizing bluegill;

(b) a deodorizing step for deodorizing bluegill;

(c) a drying step for drying the bluegill;

(d) a crushing step for crushing bluegill;

(e) a low-temperature pressing extraction step of extracting bluegill by low-temperature pressing at a temperature of 55 to 65°C; and

(f) A purification step for purifying the bluegill low-temperature press extract.

The method for producing bluegill extract of the present invention is described in detail as follows.

(a) Sterilization step

In the present invention, the method of sterilizing bluegill can utilize various fish sterilization methods known in the art, and for example, sterilization can be performed by immersing in a solution containing a carbon source and a weak acid for 6 to 8 hours.

In the present invention, there is no particular limitation on the carbon source as long as it provides sufficient carbon, but charcoal can be preferably used. The weak acid can preferably be acetic acid (CH ₃ COOH) as a component of vinegar.

In the present invention, by immersing bluegill in a solution containing a carbon source and a weak acid, it is possible to sterilize the bluegill without damaging its flesh.

In the present invention, the sterilization step may include a step of immersing in a solution containing a carbon source and a strong acid for 6 to 8 hours.

If the sterilization time is less than 6 hours, the germs and bacteria inherent in the flesh of bluegill will not be sufficiently removed, and if the sterilization time exceeds 8 hours, there may be concerns about the destruction of nutrients, including taurine, and damage to the flesh.

In the present invention, the bluegill in the sterilization step is not particularly limited, and may include, for example, the flesh, skin, entrails, and head of the bluegill as a deboned bluegill.

(b) Deodorization stage

In the present invention, the characteristic fishy smell of bluegill can be removed through the deodorizing step.

In the present invention, the deodorizing step can be performed by immersing bluegill in at least one selected from the group consisting of fruit juice and alcohol.

In the present invention, the deodorizing step may include a step of immersing the bluegill in a solution in which lemon juice or orange juice is mixed with one or more alcohols selected from the group consisting of cheongju, soju, and wine.

The acid in the fruit juice neutralizes the fishy smell of bluegill, weakening the odor, and as the alcohol component evaporates, it also evaporates the fishy smell, effectively removing the fishy smell.

(c) Drying stage

In the present invention, the drying step may include a step of drying the bluegill with hot air at 60 to 70° C.

If it is below 60℃, there may be a problem of raw material oxidation during the drying stage, and if it exceeds 70℃, there may be a problem of active ingredient destruction.

(d) crushing stage

In the present invention, the crushing method can utilize various crushing methods known in the art, and for example, the bluegill can be crushed using a crusher.

In the present invention, the particle size of the bluegill powder that has undergone the pulverization step is not particularly limited, but may be 0.01 mm to 1.0 mm, for example, 0.04 mm to 0.08 mm.

(e) Low temperature pressing extraction stage

In the present invention, the low temperature pressing extraction step may be performed at a temperature of 55 to 65°C.

If it is below 55℃, there may be a problem of very low yield, and if it exceeds 65℃, there may be a problem of destruction of effective ingredients.

In the present invention, the low-temperature pressing extraction step may be performed at a pressure of 600 to 650 kgf/cm ² .

If it is less than 600 kgf/ ^cm2 , there may be a problem of inefficiency in the extraction amount per time, and if it exceeds 650 kgf/ ^cm2 , the change in the extraction amount per time is not significant and there may be a problem of overload of the extraction equipment.

In the present invention, the low temperature pressing extraction step may be performed for 0.5 to 1 hour.

If it is less than 0.5 hours, there may be a problem of not being able to secure sufficient extracts, and if it exceeds 1 hour, there may be a problem of reduced production competitiveness due to no change in the amount of extraction over time.

In the present invention, the method for producing a bluegill extract includes a low-temperature pressing extraction step, thereby producing a bluegill extract containing 70 to 90% by volume of high-purity unsaturated fatty acids.

In the present invention, the method for producing bluegill extract can achieve the effect of reducing cost by including a low-temperature pressing extraction step.

(f) Purification step

In the present invention, the purification step may include deacidification, degumming and decolorization steps.

The deoxidation step is a process of removing free fatty acids, etc., during the maintenance process, and is also called alkaline refining. When free fatty acids are neutralized, soap particles are created, and since soap particles absorb impurities such as pigments and precipitate, the neutral oil floating on the top layer has a lower acid value and a clearer color.

In the present invention, the deoxidation step may include a step of adding potassium hydroxide to bluegill cold-pressed extracted oil and heating it.

In the present invention, potassium hydroxide may be added in an amount of 0.5 to 2% (v/v) relative to the total volume of bluegill cold-pressed extracted oil.

In the present invention, ethanol may be added in an amount of 10 to 20% (v/v) relative to the total volume of bluegill cold-pressed extracted oil.

In the present invention, heating may be performed at a temperature of 50 to 80° C., 50 to 70° C., 60 to 80° C., or 60 to 80° C.

In the present invention, free fatty acids and glycerol can be removed through the deoxidation step.

In the present invention, the deoxidation step may include a process for reducing saturated fatty acids.

In the present invention, the saturated fatty acid reduction process may include a step of adding potassium hydroxide and ethanol and heating and stirring.

In the present invention, the deoxidation step may include a step of removing impurities after centrifugation and collecting the supernatant.

The degumming step refers to the process of removing protein breakdown products and viscous substances that are abundantly contained in fish oil, etc. For example, lecithin, keparin, etc. swell in water and do not dissolve in oil, but rather precipitate out, so they are separated from the oil using a centrifuge.

In the present invention, the degumming step may include a step of adding distilled water to the supernatant collected in the deoxidation step and heating and stirring it.

In the present invention, distilled water may be added in an amount of 10 to 30% (v/v) relative to the total volume of the supernatant collected in the deoxidation step.

In the present invention, heating and stirring may be performed at a temperature of 50 to 80°C, 50 to 70°C, 60 to 80°C, or 60 to 80°C.

In the present invention, the degumming step may include a step of removing impurities after centrifugation and collecting the supernatant.

In the present invention, the degumming step can be repeated 2 to 3 times.

The decolorization step is the process of adding a decolorant to the separated oil, reducing pressure, heating and stirring, and then filtering using a filter press, filter paper, and centrifuge to separate the oil and decolorant.

In the present invention, the decolorization step may include a step of adding activated carbon and activated clay to the supernatant collected in the decolorization step and heating and stirring the same.

In the present invention, activated carbon and activated clay may be added in an amount of 10 to 20% (v/v) relative to the total volume of the supernatant collected in the degumming step.

In the present invention, heating and stirring may be performed at a temperature of 50 to 80°C, 50 to 70°C, 60 to 80°C, or 60 to 80°C.

In the present invention, the heating and stirring may be performed for 30 to 90 minutes, 30 to 80 minutes, 40 to 90 minutes, or 40 to 80 minutes.

In the present invention, the decolorization step may include a step of removing impurities after centrifugation and collecting the supernatant.

In the present invention, the degumming step may include a step of filtering oil stirred with activated carbon and activated clay through a mesh, centrifuging to remove impurities, and collecting the supernatant.

In the present invention, the method for producing a bluegill extract includes deacidification, degumming, and decolorization steps, thereby producing a bluegill extract containing 70 to 90% by volume, 70 to 80% by volume, or 80 to 90% by volume of a high-purity unsaturated fatty acid.

The present invention relates to an antioxidant and anti-inflammatory composition comprising a bluegill extract and a method for producing a bluegill extract. The bluegill extract exhibits excellent antioxidant effects through DPPH radical scavenging activity and ABTS scavenging activity, and has excellent anti-inflammatory effects by inhibiting the production of NO by LPS. Therefore, the composition of the present invention comprising a bluegill extract can be usefully used as an antioxidant and anti-inflammatory composition.

Figure 1 is a graph showing the results of a cytotoxicity test of a bluegill extract according to one embodiment of the present invention.
Figure 2 shows the results of analyzing the oxygen radical scavenging ability of a bluegill extract according to one embodiment of the present invention through a DPPH assay.
Figure 3 shows the results of analyzing the antioxidant capacity of a bluegill extract according to one embodiment of the present invention using an ABTS assay.
Figure 4a shows the results of analyzing the anti-inflammatory effect of a bluegill extract according to one embodiment of the present invention in the absence of LPS treatment using a NO assay.
Figure 4b shows the results of analyzing the anti-inflammatory effect of a bluegill extract according to one embodiment of the present invention when treated with LPS using a NO assay.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of bluegill extract 1 (bluegill oil)

1-1. Bluegill Sterilization Treatment

Bluegills were sterilized by soaking them in a mixture of carbonaceous material (charcoal) and acetic acid (CH ₃ COOH) for 6 hours.

1-2. Deodorizing treatment

After sterilization, bluegill was cleaned and soaked in a liquid containing lemon containing citral (C ₁₀ H ₁₆ O) and sake containing ethyl alcohol (C ₂ H ₅ OH) for 6 to 8 hours. Freshwater fish such as bluegill have a fishy smell unique to freshwater fish due to a mixture of piperidine and acetaldehyde. Trimethylamine and ammonia, the main culprits of the fishy smell, dissolve in water and can solve the fishy smell problem to some extent, but not perfectly. Therefore, acid (lemon) was added to neutralize the fishy smell component and weaken the smell, and ethyl alcohol (cheongju) was treated so that when the ethyl alcohol component evaporates, it evaporates together with the fishy smell component, thereby enhancing the effect of removing the fishy smell.

1-3. Dismantling and hot air drying

By-products (head, entrails, tail, skin, and bones) other than the bluegill flesh were separated and dried with hot air at 55°C.

1-4. Crushing

The by-products, from which moisture was removed through dismantling and hot air drying, were crushed into 60 mesh.

1-5. Low temperature pressing extraction

Using oil press equipment [Twin-pod Oil Press (safe type)], oil was extracted under the conditions of 611.8 kgf/ ^cm2 , 60℃, and 1 hour, and primary impurities were removed through a 200 mesh screen to obtain bluegill low-temperature pressing extracted oil.

1-6. Refining

The bluegill cold-pressed extract oil was centrifuged at 3000 rpm for 15 minutes to remove the primary sediment residue. Afterwards, it was purified through deoxidation, degumming, and decolorization processes and used as a bluegill extract. 105 g of refined oil was obtained per 200 g of cold-pressed extract oil, and the yield was measured to be 52.5%. The specific processes of deoxidation, degumming, and decolorization are as follows.

1-6-1. Deacidification

200 g of bluegill cold-pressed extract oil was heated at 60°C for 1 hour with 0.5 to 2% (v/v) potassium hydroxide and 10 to 20% (v/v) ethanol to remove free fatty acids and glycerol.

After cooling to room temperature (approximately 25°C), the solution was centrifuged at 2,500 rpm for 15 minutes to remove impurities and collect the supernatant.

1-6-2. De-staining

10 to 20% (w/w) of distilled water at 80°C was added to the collected supernatant, and the mixture was heated and stirred at 60°C for 20 minutes. After that, the mixture was centrifuged at 2,500 rpm for 15 minutes to remove impurities and collect the supernatant. This process was repeated 2 to 3 times.

1-6-3. Bleaching

5-10% (w/w) of activated carbon and activated clay were added to the collected supernatant, and heating and stirring were performed at 60 ℃ for 30-60 minutes. After that, centrifugation was performed at 2,500 rpm for 15 minutes, impurities were removed, and the supernatant was collected. The oil stirred with activated carbon and activated clay was filtered through a 200 mesh screen, centrifugation was performed at 3,000 rpm for 15 minutes, impurities were removed, and the supernatant was collected. After filtering the oil through filter paper, the final purified bluegill oil was obtained, which was used as a bluegill extract.

The pH and acid value of the final refined bluegill oil (hereinafter, bluegill extract 1) were measured to be 7.3 to 7.6 (neutral) and 0 to 0.5 (fish oil 3 or less), respectively, which do not exceed the standards described in the Item-by-Item Standards and Specifications for Single Feed (related to Article 8, Paragraph 1) and Article 5. Standards and Specifications for Each Food of the Food and Food Additives Codex of the Ministry of Food and Drug Safety. The specific components and contents of unsaturated fatty acids contained in bluegill oil were analyzed and are shown in Table 1.

Unsaturated fatty acids Content (vol%) Lauric Acid Methyl Ester (C12:0) 0.324 Tridecanoic Acid Methyl Ester (C13:0) 0.825 Myristic Acid Methyl Ester (C14:0) 2.896 Myristoleic Acid Methyl Ester (C14:1) 0.547 Pentadecanoic Acid Methyl Ester (C15:0) 0.654 cis-10-pentadecenoic methyl ester 0.272 Palmitic Acid Methyl Ester (C16:0) 19.866 Palmitoleic Acid Methyl Ester (C16:1) 12.071 Heptadecanoic Acid Methyl Ester (C17:0) 1.243 Stearic Acid Methyl Ester (C18:0) 3.937 Oleic Acid Methyl Ester (C18:1n9c) 18.686 Linoleic Acid Methyl Ester (C18:2n6c) 6.353 Linolenic Acid Methyl Ester (C18:3n3) 4.912 cis-11,14-Eicosadienoic Acid Methyl Ester (C20:2) 2.921 Erucic Acid Methyl Ester (C22:1n9) 1.744 cis-13,16-Docosadienoic Acid Methyl Ester (C22:2) 2.846 Lignoceric Acid Methyl Ester (C24:0) 4.544 Unknown fatty acids 15.358 Total (vol%) 100.000

Example 2. Preparation of bluegill extract 2 (bluegill protein)

Bluegill was sterilized, deodorized, disassembled, hot-air dried, and ground as in Example 1. Four times the weight of the ground bluegill was added with distilled water, and heating and stirring were performed at 80°C for 1 hour. The hot water extract was filtered through a 200 mesh, placed in a tube, and centrifuged at 3000 rpm for 15 minutes. The supernatant was collected, treated with 1% (v/v) of the enzyme alcalase, and then heating and stirring were performed at 60°C for 30 minutes. In order to inactivate the enzyme reaction, heating and stirring were performed at 80°C for 20 minutes, followed by freeze-drying, and then bluegill protein was manufactured as a powder.

Experimental Example 1. Cytotoxicity Test (MTT Assay)

The cytotoxicity of the bluegill extracts of Examples 1 and 2 was tested by MTT assay and is shown in Fig. 1. As can be seen from the results in Fig. 1, bluegill protein and bluegill oil showed no cytotoxicity at all concentrations.

Experimental Example 2. Evaluation of Antioxidant Activity

2-1. Evaluation of active oxygen scavenging activity (DPPH assay)

0.25 mM DPPH was dissolved in ethanol to make a final concentration of 0.0019 g/20 mL, and filtered through a 0.22 μm filter syringe. The control group (standard) was prepared as stocks of 1 μM, 5 μM, 10 μM, and 50 μM, respectively, and the bluegill proteins were prepared as 5 μg, 20 μg, 100 μg, and 200 μg, respectively, and the bluegill oil was prepared as dilutions of 1% (v/v), 2% (v/v), 5% (v/v), and 10% (v/v). 50 μL of DPPH reagent was dispensed into the tube, and equal amounts of the control group and each bluegill extract sample were dispensed. 50 μL of distilled water was dispensed to remove the retained color of the samples, and then equal amounts of each extract sample were dispensed. After vortexing at 250 rpm, the mixture was reacted for 60 minutes at room temperature (approximately 25°C) and dispensed into 96-well plates in 5 replicates of 50 μl each. The absorbance was measured at 450 nm using an ELISA reader, and the results are shown in Figure 2.

As can be seen from the results in Fig. 2, bluegill protein and oil showed high radical scavenging ability, confirming that they have an excellent effect in removing active oxygen.

2-2. Antioxidant activity evaluation (ABTS assay)

200 mM ABTS and 100 mM potassium persulfate were dissolved in distilled water, mixed, and reacted for 1 hour at room temperature (approximately 25 ℃) in a light-blocking state. The absorbance was measured at 450 nm in an ELISA reader. The extract samples were diluted with distilled water according to their concentrations and prepared. The control group (ascorbic acid) was prepared as stocks of 1 μM, 5 μM, 10 μM, and 50 μM, respectively, and the bluegill proteins were prepared as 5 μg, 20 μg, 100 μg, and 200 μg, respectively, and the bluegill oil was prepared as dilutions of 1% (v/v), 2% (v/v), 5% (v/v), and 10% (v/v). 50 μl of DPPH reagent was dispensed into tubes, and equal amounts of the control group and each bluegill extract sample were dispensed. To remove the retained color of the sample, distilled water was dispensed at 50 μl each, and then each extract sample was dispensed in an equal amount. After vortexing at 250 rpm, the mixture was reacted at room temperature (approximately 25 °C) for 20 minutes, and 50 μl each was dispensed into 96-well plates in 5 replicates. The absorbance was measured at 450 nm using an ELISA reader, and the results are shown in Fig. 3.

As can be seen from the results in Figure 3, bluegill protein and oil showed high radical scavenging ability, confirming excellent antioxidant effects.

Experimental Example 3. Evaluation of anti-inflammatory activity (NO assay)

RAW274 cells were seeded in 24-well plates and treated with diluted extract samples and LPS (100 μg/ml) in the medium at a concentration of 10% and incubated for 2 h. The supernatant was collected and the absorbance was measured at 450 nm. The anti-inflammatory activity of the bluegill extract was evaluated by a method for quantifying the amount of NO produced and secreted by immune activation using RAW274 cells. Specifically, bluegill proteins were prepared in concentrations of 5 μg, 20 μg, 100 μg, and 200 μg, respectively, and bluegill oils were prepared in dilutions of 1% (v/v), 2% (v/v), 5% (v/v), and 10% (v/v), respectively. The NO concentration was measured after 2 hours of incubation under LPS (Lipopolysaccharide)-free conditions, and the results are shown in Fig. 4a. The NO concentration was measured after 2 hours of incubation with LPS (100 μg/ml), and the results are shown in Fig. 4b.

As can be seen from the results in Figures 4a and 4b, bluegill oil effectively inhibited NO production in both LPS-untreated and -treated conditions, confirming that the oil extract exhibited an anti-inflammatory effect.

Claims (8)

An antioxidant or anti-inflammatory composition comprising a bluegill extract. An antioxidant or anti-inflammatory composition in claim 1, wherein the bluegill extract is an extract including bluegill oil obtained by cold-pressing bluegill at 55 to 65°C. An antioxidant or anti-inflammatory composition in claim 1, wherein the bluegill extract is an extract containing bluegill protein obtained by hot water extraction and enzyme treatment. An antioxidant or anti-inflammatory composition according to claim 1, wherein the composition is a cosmetic composition. An antioxidant or anti-inflammatory composition in claim 1, wherein the composition is a food composition. An antioxidant or anti-inflammatory composition in claim 1, wherein the composition is an animal feed composition. A method for preparing bluegill extract comprising the following steps:
(a) a sterilization step for sterilizing bluegill;
(b) a deodorizing step for deodorizing bluegill;
(c) a drying step for drying the bluegill;
(d) a crushing step for crushing bluegill;
(e) a low-temperature pressing extraction step of extracting bluegill by low-temperature pressing at a temperature of 55 to 65°C; and
(f) A purification step for purifying the bluegill low-temperature press extract. A manufacturing method in claim 7, wherein the purification step includes deoxidation, degumming and decolorization steps.