WO2013172525A1

WO2013172525A1 - Cosmetics, pharmaceuticals, and food composition containing pieces or extract from fish eyes

Info

Publication number: WO2013172525A1
Application number: PCT/KR2012/010549
Authority: WO
Inventors: 박성민; 박성노; 서일호; 박지영; 윤미영; 최수연
Original assignee: 주식회사 코씨드바이오팜; 주식회사 뉴메디온
Priority date: 2012-05-16
Filing date: 2012-12-06
Publication date: 2013-11-21
Also published as: JP2015523333A; CN104411296A

Abstract

The present invention relates to a multifunctional cosmetic composition containing pieces or an extract from fish eyes as an active ingredient, which has good skin whitening effects and good skin resiliency improving effects, and has good skin moisturizing effects and cell activation effects, so as to exhibit excellent skin-regenerating and wound healing effects as well as excellent irritation relief effects. Also, the present invention relates to a composition for preventing and treating arthritis, which includes pieces or an extract from fish eyes as an active ingredient. The composition of the present invention promotes immune tolerance in the intestinal immune system without having any side effects so as to prevent and treat arthritis by effectively controlling autoimmune reactions and infections which particularly occur in joints. In addition, the present invention relates to a pharmaceutical composition for treating dry eye, which includes an extract from fish eyes as an active ingredient. The present invention uses fish eye extract to effectively treat eye disorders, such as conjunctival epithelium disorder, without the traditional side effects from the repeated administration of anti-inflammatory drugs.

Description

Cosmetic, pharmaceutical and food compositions containing shreds or extracts of fish eye

The present invention relates to a cosmetic composition containing a lysate or extract of fish eye, and more particularly, to a multifunctional cosmetic composition containing a lysate or extract of fish eye.

In addition, the present invention relates to a pharmaceutical composition and food composition for preventing or treating arthritis, including the lysate or extract of fish eye as an active ingredient.

The present invention also relates to a pharmaceutical composition for treating dry eye syndrome comprising fish ocular extract as an active ingredient.

Bluefin tuna (Thunnus thynnus (Linnaeus)) is a saltwater fish of the perch mackerel family. It is called tuna in Gangwon-do. The species inhabiting the North Atlantic grows up to 3m in length and 560kg in weight. The body is fat, close to fusiform, and the height is slightly higher. The snout is long and pointed and its mouth is large. The back of the body is dark blue, and the center of the body and the abdomen are silver gray with several narrow white horizontal bands and round patterns. When they are young, narrow horizontal bands and rounded patterns fade out and gradually disappear as they grow up. Small round scales cover the entire body. It looks similar to whitefin tuna, but the short pectoral fin is bluefin tuna. Swim just below sea level and sometimes appear near the coast. It moves north in spring and summer and south in autumn. The group usually eats anchovies, saury, and herring, and eats shrimp, crabs, squids, and jellyfish. The spawning season is from April to June near the coast of Taiwan and August from the east coast of Korea. The main spawning grounds are the southern part of the East Sea and the northern part of Taiwan. Bluefin tuna is rich in protein and amino acids, unsaturated fatty acids omega-3, and contains a large amount of trace elements such as vitamins and selenium. Bluefin tuna is known to contain omega-3, protein, and water. have.

Mackerel (Scomber japonicus) is a saltwater fish of the perch mackerel family, and the back side is dark blue and the belly side is silver white. The body is long and fusiform and slightly flanked. Eyes are large, oily eyelids are well developed, and pupils are exposed. Between the eyes is flat The back end of the upper jaw reaches the middle of the pupil. The dorsal fin is two distant, similar in basal length but higher in first dorsal fin. Pectoral fin located at center of body side and relatively small. Dorsal fin in symmetrical position with second dorsal fin. Dorsal fin and posterior dorsal fins with five cut fins. Tail caudal. The caudal fin is well developed crotch. The first dorsal fin has the longest second spine. The back of the body is dark blue and silver white from the center to the abdomen. Blue-black wave pattern is distributed to the side line on the back of body. Dorsal fin transparent but black vesicles interspersed, dark, pectoral fin base white, upper edge of base and black late. Badge fin and dorsal fin colorless and transparent, caudal fin gray, but outer edge black. It grows up to 50 cm in length, but typically ranges from 30 cm.

Two species of mackerel are known in Korea, mackerel and hammered mackerel. Hammer mackerel is well distinguished by the round dark blue pattern along the center of the body side, but individual variation is observed between the mackerel and hammer mackerel. On the other hand, hammer mackerel is distinguished from mackerel because it has the longest 3 ~ 4 spines of first dorsal fin. The habitat depth is 0 to 300 m. It is a swollen fish species and lives in the middle layer within 300m from the surface or surface layer. If they grow more than 3cm, they live in clusters by size. In the Northeast Pacific, they can also flock together with other species. It is distributed in Korea, tropical and temperate seas of the entire ocean. It is collected in the East China Sea. Spawning takes place at a water temperature of 15 to 20 ° C, with slight differences depending on the region. Seasonal appeasement, species in the northern hemisphere migrate to the north during summer when the water temperature rises and to the south during winter. It feeds on copepods, crustaceans, fish, and squids, and competes with other species that live in colonies. Mackerel is rich in DHA and EPA, a type of omega-3, and is known to be rich in calcium, iron, potassium, vitamin B2 and vitamin D, and rich in protein and amino acids.

Thus, although bluefin tuna or mackerel is rich in various nutrients, there are no examples of using the eye (eye) of such bluefin tuna or mackerel, especially bluefin tuna or mackerel as a raw material for cosmetics, pharmaceuticals or food compositions.

On the other hand, recently, cosmetics based on natural materials have been in the spotlight by consumers for reasons of human stability and skin irritation, and accordingly, various researches on natural products have been conducted as cosmetic raw materials.

Accordingly, research is being conducted to use fish as a cosmetic raw material. In this regard, Korean Patent Application Publication No. 10-1999-0033721 (a method for extracting water-soluble COLLAGEN from fish and a method for preparing functional COLLAGEN cosmetics and COLLAGEN fermented beverages) Disclosed is a method for extracting and separating collagen from the skin, hair, bones, internal organs, etc., and a method of extracting gelatin from the skin in the Republic of Korea Patent No. 10-0760875 (cosmetic composition for inhibiting melanin production) In addition, Korean Patent No. 10-0986603 (DNA polymer fragment composite separated from fish semen or eggs and a method of preparing the same) discloses a cosmetic composition comprising a DNA fragment mixture separated from fish semen or eggs. However, there is no example of using the eye of the subclass (eyeball) as a cosmetic raw material yet.

Arthritis, on the other hand, is a chronic disease that affects 5% of the Korean population, which causes inflammation of the synovial joints of the joints and causes swelling and pain. Arthritis is a progressive disease that causes joint deformities and disorders that, if not treated, continues to worsen and have serious consequences.

The direct cause of arthritis is not yet clear, and for the treatment of steroids such as cortisone and other corticosteroids, nonsteroidal anti-inflammatory agents such as aspirin, pyroxicam and indomethacin, chloroquinones and Various therapeutic agents are commonly used, including antirheumatic agents such as D-phenicylamine, gout inhibitors such as colchicine, and immunosuppressive agents such as cyclophosphamide, azathioprine, methotrexate, and levamisol. However, these chemotherapeutic agents do not have a fundamental therapeutic effect, and in the case of steroid hormones, their use is limited due to side effects. In addition, aspirin and betazoline, which are widely used as drugs for alleviating joint pain or removing inflammation, have a fatal effect on the stomach of the person, and thus it is impossible to continuously take the amount necessary to treat arthritis.

Existing chemotherapeutic agents have drawbacks such as side effects that make long-term use of drugs difficult, and decreased anti-inflammatory effects in long-term use. Currently, indomethacin, furpenic acid, and nonsteroidal anti-inflammatory agents having excellent analgesic activity are used.

Therefore, it is desired to develop an arthritis therapeutic agent that solves these problems and exhibits effects on inflammatory symptoms and pain, and it is very important to develop a drug having fewer side effects since the arthritis therapeutic drug requires long-term administration. In addition, some of the existing drugs may be administered by intravenous injection or intraperitoneal injection, in which case it is not only cumbersome, but also allergic, shock and hygiene problems may occur, so it is necessary to take a simple and safe treatment.

To date, most health foods for the treatment of arthritis have cartilage components as the main ingredient. Korean Patent Laid-Open No. 2001-0018321 discloses a health food composition for rheumatoid arthritis patients, and Korean Patent Laid-Open No. 2005-0078080 discloses a pharmaceutical composition for treating arthritis and a manufacturing method thereof, and a Korean patent application No. 10-2003-00433119 describes a dietary supplement composition for treating degenerative arthritis comprising 40-50% of glucosamine, 30% of mucopolysaccharide protein, vitamin C, dew, Chinese quince, tofu and shark cartilage extract powder. have. However, there have been no reports on the use of fish eye debris or extracts in connection with the prevention or treatment of arthritis.

Accordingly, the present inventors have confirmed that fish eye debris or extract reduces redness and swelling in collagen-induced animal models and reduces blood levels of cytokine molecules that have been found to cause arthritis, thereby preventing or treating arthritis. The present invention has been completed by revealing that it can be usefully used.

On the other hand, prolonged use of the eyes, such as prolonged reading, computer use, driving or watching television, reduces the blink rate of the eyelids, and reducing the blinking covering the eyes with tears causes worsening ophthalmic symptoms.

Ophthalmology-related diseases continue to increase because of the aging population, the improvement of portable terminal performance, the development of communication technology, and the growth of the cultural industry. .

Dry eye syndrome refers to diseases such as conjunctival dryness, hypotony, and dry keratoconjunctivitis in consultation, but broadly means all the symptoms in which the conjunctiva is ideally dried. As such, dry eye syndrome is thought to be widespread in concept and cause, but in many cases, the cause is unknown, so it is a disease of the surface of the eye, including diseases called dry eye syndrome, rather than a single disease.

Currently, dry eye is defined as an abnormal state of tear quantity and quality regardless of keratoconjunctivitis disorder. By definition, dry eye categories include diseases such as hypotony, tear deficiency, dry eye syndrome, Sjogren's syndrome, dry keratoconjunctivitis, Stevens-Johnson syndrome, ocular swelling, blepharitis, blepharophagia, and perceptual nerve palsy. do.

The cornea and conjunctiva of the eye tissues are epithelial tissues exposed to the outside and are easily damaged by external factors. The method of healing the damage of local area caused by various external factors is pharmacologically acting on the affected area, and aims to prevent the secondary damage of the tissue by inflammation and to increase the rate of tissue regeneration. The causes of inflammation include pathogenic microbial infections, chemical infiltration of substances causing inflammation, allergies, irritable autoimmune diseases, and physical wounds.

In many cases with dry eye, tears in either the oily, aqueous or mucin layers cause keratoconjunctivitis disorders. In particular, when tears are scarce in the mucin layer, corneal damage is severe, including corneal epithelial erosion resulting from corneal epithelial cell damage, corneal epithelial disorders, as well as corneal ulcers (eg, ulcers in the corneal stroma) and infectious eye diseases. Cause easily. In some cases, corneal transplantation is required.

In addition, soft contact lenses can cause dry eye problems. Although soft contact lenses are water-soluble, it is difficult to distribute a water layer having a sufficient thickness on the lens surface, so that the dispersion of the lipid layer deteriorates, and the evaporation of tears on the lens may be a cause of corneal epithelial disorder.

However, there have been no reports on the use of fish eye extracts for the treatment of dry eye syndrome. Thus, the present inventors have completed the present invention by revealing that the fish eye extract is effective in the treatment of corneal epithelial cell disorders by reducing the water evaporation of the eye, and can be useful in the treatment of dry eye syndrome.

It is an object of the present invention to provide a debris or extract of fish eye which is applicable to skin cosmetics and is harmless to the human body and has excellent safety.

Another object of the present invention is to provide a cosmetic composition having excellent skin whitening effect, skin moisturizing effect, and skin elasticity improving effect by using crushed or extract of fish eye.

In addition, an object of the present invention is to provide a cosmetic composition having excellent cell activation effect by using the lysate or extract of fish eye, excellent skin regeneration and wound healing effect, and also very excellent in stimulating effect.

In addition, an object of the present invention is to provide a pharmaceutical composition and functional food for preventing or treating arthritis, including fish eye debris or extract.

It is also an object of the present invention to provide a pharmaceutical composition for treating dry eye syndrome comprising fish ocular extract.

As used herein, the term "fish" refers to vertebrates that have gills that live in water, and the fishes include, for example, skates, cod, mackerel, dome, smelt, salmon, flock, seatail, devil, great white shark, defense, pollock , Stilt, carp, crucian carp, herring, marlin, red snapper, bluefin tuna, mullet, mackerel, fishfish, sea bream, saury, sunfish, black sea bream, blackfish, stingray, bes, loach, catfish, crabfish, mintae, hwangseongdae, swordfish At least one fish selected from the group consisting of yellowfin tuna, blue shark, reference group, abbreviation, participant, larvae, larvae, zodiac rockfish, sardine, horse mackerel, zodiac, mackerel fish, jade fish, eel, bottlefish, and fish. Most preferably, the fish that can be used in the present invention are bluefin tuna or mackerel.

In the present specification, the term "eyeball" refers to an organ that collects visual information, converts it into electrochemical information, and delivers it to the brain through a passage called the optic nerve. As shown below, the fish's eye has the outermost sclera (existing as the cornea in front of the lens), the choroid inside, and the retina inside, and the lens and iris in the front. The front is filled with fluid, and the back is filled with glass fluid. Others include scythes (similar to human ciliary bodies), muscles, and pigments.

[Eye Structure of Fish]

And as used herein, the term "crushed product" refers to the result of crushing the fish eye of the present invention. Specifically, the eyeballs are removed from the head of the fish, the muscle tissues attached to the sclera of the eyeballs are prepared, and cleanly separated eyes are prepared. The material is exposed to the outside and crushed using a mixer or homogenizer which is generally used in the art. At this time, preferably it is preferably treated homogeneously in the size of 1㎛ to 5mm easily absorbed into the human body. In addition, the fish eye crushed product of the present invention may be a filtrate obtained by filtering the fish eye crushed homogeneously as described above.

In order to achieve the above object, the first embodiment of the present invention provides a cosmetic composition characterized in that it contains a lysate or extract of fish eye as an active ingredient.

Hereinafter will be described in detail with respect to the shreds or extracts of the fish eye of the present invention, and the cosmetic composition containing the same.

The present invention is characterized by using the fish's eye, that is, the eye as an active ingredient of the cosmetic composition. As described above, the present invention uses the eye of the bluefin tuna or mackerel that is not effectively utilized as waste, and thus, there is an advantage that it is possible to prevent secondary environmental problems as well as utilizing waste resources.

Here, the eye of the fish is preferably using the eye (eye) of at least one type of fish of bluefin tuna or mackerel, more preferably frozen fish eye obtained by quenching fresh bluefin tuna or mackerel and then separated from meat Eyeball).

The present invention contains the lysate of the fish eye as an active ingredient, wherein the method of obtaining the lysate of the fish eye is not particularly limited, and a method commonly used in the art to which the present invention pertains may be used. For example, the frozen fish eye may be crushed by using a crusher, for example, a mixer or homogenizer, and is preferably homogeneously processed to a size of 1 μm to 5 mm that is easily absorbed into the human body. It is good to be.

In addition, the present invention contains the extract of the fish eye as an active ingredient, wherein the method of obtaining the fish eye extract is not particularly limited, and various extraction methods commonly used in the art to which the present invention pertains may be used. For example, purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, hexane can be extracted alone or in combination of two or more of them as a solvent for extraction. .

In addition, the extract of the fish eye of the present invention includes not only the extract by the aforementioned extraction solvent, but also extract extracted by other methods. Extraction by, for example, ultrahigh pressure extraction; Separation using ultrafiltration membranes with constant molecular weight cut-off values; Fractions obtained through various purification methods additionally performed, such as by chromatography prepared for separation according to size, charge, hydrophobicity or affinity, are included in the extract of fish eye of the present invention. .

In the present invention, when the fish eye is subjected to ultra high pressure extraction using an ultra high pressure extraction method, the treatment is preferably performed at a pressure of 100 to 3000 MPa and a temperature of 40 to 90 ° C for 5 to 60 minutes, and more preferably a pressure of 500 to 1000 MPa and a temperature of 40 to It is good to process it for 10 to 30 minutes at 60 degreeC. In addition, the pH at the time of ultrahigh pressure extraction is not limited to a specific pH, but preferably, the pH of the water inside the ultrahigh pressure is lowered to 1 to 6 and treated. In addition, the present invention using the ultra-high pressure treatment of the eye extract of bluefin tuna or mackerel using an extraction solvent selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, hexane and mixtures thereof After reflux extraction and chilling, the filtered filtrate is transferred to a concentration tank, preferably concentrated under reduced pressure and freeze-dried at 50 ° C or lower. The present invention can be prepared by using one or more solvents selected from purified water, ethanol, butylene glycol, propylene glycol so as to contain 0.001 ~ 70.0% by weight of the vacuum concentrate and lyophilized thus obtained.

In addition, the extract of the fish eye of the present invention is a concentrate obtained by partially or fully concentrating the extract, or extract extract prepared by drying the concentrate again and the chemical itself exhibiting the main effect contained in the extract itself. Include.

Thus obtained lysate or extract of fish eye of the present invention, melanin production inhibitory effect, anti-collagenase / gelatinase activating effect, MMP-1 expression inhibitory effect after ultraviolet irradiation, cytotoxicity relieving effect by ultraviolet irradiation, skin moisturizing Excellent effect, skin elasticity effect, irritation relief effect, wound healing effect can be used as a multifunctional cosmetic composition showing a complex combination of skin whitening, skin elasticity or skin moisturizing effect.

The cosmetic composition of the present invention preferably contains 0.0001 to 90% by weight of the fish eye shreds or extracts, based on the total weight of the composition, when less than 0.0001% by weight is difficult to expect the effect, more than 90% by weight When added, no marked increase in efficacy effect is seen.

In addition, the cosmetic composition of the present invention, in addition to the crushed products or extracts of the fish eye, preferably containing other components and the like that can give a synergistic effect to the main effect within the range that does not impair the main effect of the present invention. It's okay. For example, it may further contain an anti-aging component, an anti-wrinkle component, a whitening component, a moisturizing component and an antibacterial component.

In addition, the cosmetic composition of the present invention is an auxiliary agent commonly used in the cosmetic field such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants, dyes, etc. It may contain. The amounts of these various adjuvants are amounts commonly used in the art, such as 0.0001 to 30% by weight relative to the total weight of the composition. In any case, the adjuvant and its proportions will be selected so as not to adversely affect the desirable properties of the cosmetic composition according to the invention.

In addition, the formulation of the cosmetic composition of the present invention is not limited to a specific kind, for example, in a lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) and water-in-oil (W / O) type Any one selected from the basic cosmetic formulation consisting of oil-in-water and oil-in-water makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencils. It may have an appearance. It may also be applied to the skin, optionally in the form of an aerosol, or may be in the form of a solid such as a stick. It may be used as a skin care product and / or makeup product.

The cosmetic composition of the present invention prepared as described above has a very good skin whitening effect, skin elasticity improvement effect, skin moisturizing effect, excellent cell activation effect and excellent skin regeneration and wound healing effect and stimulating alleviation effect. It can be used as.

On the other hand, according to a second embodiment of the present invention, the present invention provides a composition for the prevention and treatment of arthritis comprising fish eye lysate or extract as an active ingredient.

The present inventors have tried to develop a composition for preventing and treating arthritis having excellent efficacy of preventing arthritis and greatly reducing side effects. As a result, the administration of fish eye debris or extract in the onset of arthritis greatly increases the immune tolerance, significantly reducing the arthritis-specific inflammatory response, thereby eventually achieving arthritis prevention and treatment efficacy. Confirmed.

In the composition of the present invention, the basic active ingredient is a fish eye debris or extract.

Fish eye shreds or extracts of the active ingredient of the present invention contains a lot of vitamin A, B2, and the like, especially in the back of the eye is known to help the carbohydrate metabolism. On the other hand, the present inventors have found that fish eye debris or extract acts synergistically to increase the immune tolerance response induced by collagen. The composition of the present invention is formulated based on this novel finding. In addition, the fish eye debris or extract in the composition of the present invention serves to promote the production of anti-inflammatory cytokines such as IL-10 and TGF-β.

In the present invention, the fish eye debris may be used by crushing the frozen fish eye using a crusher, for example, a mixer or homogenizer, preferably homogeneous in the size of 1 ㎛ to 5 mm that is easily absorbed inside the human body. Should be handled. Fish eye debris in the present invention by itself can significantly improve arthritis specific inflammatory response to eventually achieve the prevention and treatment of arthritis, can also be used in the form of extracts extracted active ingredients to increase the prevention and treatment efficacy.

In the present invention, the fish eye extract is used as a polar solvent of a lower (C1-C4) alcohol, such as water, methanol, or ethanol, 2 to 20 times, preferably about 2 to 5 times, to the fish eye shreds. The extract obtained by cold extraction, reflux cooling extraction, hot water extraction, ultrasonic extraction, ultra high pressure extraction, etc. at 10 ° C to 30 ° C extraction temperature is filtered, concentrated under reduced pressure, or lyophilized to obtain an extract available in a polar solvent. It is possible.

In the present invention, when the ultra-high pressure extraction method is used for the extraction, it is easy to elute the useful components due to the destruction of the fish eye cell tissue, and is caused by weak bonds such as hydrogen bonds, electrical bonds, and van der Waals bonds with limited energy levels. The bond can be separated and the new material is eluted, the cytotoxic material is destroyed, the important constituents can be extracted within a short time, and the extract is almost free of impurities and has the advantage of easily obtaining a high purity single component.

The ultra-high pressure extraction in the present invention is preferably treated for 5 to 60 minutes at a pressure of 100 to 3000MPa, 40 to 90 ℃ temperature, more preferably 10 to 30 minutes at a pressure of 500 to 1000 MPa, temperature 40 to 60 ℃. When the pressure condition is a low pressure condition of 100MPa or less, or when the extraction time is within 5 minutes, the ocular cell tissue destruction is insufficient, the elution efficiency of useful components is low, the pressure condition is a high pressure condition of 3000MPa or more, or extraction If the time is 60 minutes or more, the energy efficiency consumed for extraction is reduced, so it is preferable to extract in the above range, and 40 to 90 ° C. temperature conditions for effectively removing weak bonds such as hydrogen bonds, electrical bonds, and van der Waals bonds. Preference is given to performing at. In addition, the pH at the time of ultrahigh pressure extraction is not limited to a specific pH, but preferably, the pH of the water inside the ultrahigh pressure is lowered to 1 to 6 and treated. When the pH is adjusted within the above range, the dissolution efficiency of the useful ingredient is increased.

In addition, the present invention is subjected to reflux extraction of the ocular extract of the fish subjected to the ultra-high pressure treatment using an extraction solvent selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, hexane and mixtures thereof. After cooling, the filtered filtrate is most preferably concentrated under reduced pressure and freeze-dried at 50 占 폚 or lower.

In addition, the present invention can be prepared by using one or more solvents selected from purified water, ethanol, butylene glycol, propylene glycol so as to contain 0.001 ~ 70.0% by weight of the vacuum concentrate and the freeze-dried product thus obtained.

Fish ocular lysates or extracts of the invention act as autoantigens that induce an immune tolerance response. Arthritis has a wide variety of autoantigens involved in immune response, making it impossible to develop specific therapeutic agents. The core principle of the present invention is to take advantage of immune tolerance that occurs in the Gut Associated Lymphoid Tissue (GALT). Immune tolerance refers to the selective immune suppression response of the immune system (GALT) under the small intestine's epithelial cells to substances delivered orally.

According to a preferred embodiment of the present invention, the composition of the present invention may further comprise glucosamine, chondroitin or a combination thereof. Both glucosamine and chondroitin are cartilage components. Thus, the use of other cartilage components (glucosamine and chondroitin), which can act as autoantigens in addition to fish eye debris or extracts, can induce a better immune tolerance response. Glucosamine also acts to promote cartilage formation and regeneration. Chondroitin not only induces an immune tolerant response but also protects cartilage by inhibiting enzymes that destroy cartilage.

The arthritis prophylactic or therapeutic composition of the present invention may be prepared as a pharmaceutical composition, in which case it includes a pharmaceutically acceptable carrier in addition to the above components as an active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention is administered orally because it must exert an effect by inducing an immune tolerance response.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.01 to 2000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention is prepared in unit dose form by being formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by one of ordinary skill in the art. Or may be prepared by incorporating into a large volume container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The arthritis prophylactic or therapeutic composition of the present invention may be prepared as a food, in particular a functional food composition. Functional food compositions of the present invention include ingredients that are commonly added in the manufacture of food, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, a flavoring agent or a natural carbohydrate may be included as an additional ingredient in addition to the fish eye debris or extract as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

Given the ease of access to food, the food of the present invention is very useful for the treatment or prevention of arthritis.

Herein, the food composition of the present invention preferably contains 0.0001 to 90% by weight of the fish eye shreds or extracts with respect to the total weight of the composition, but when added to less than 0.0001% by weight, the effect is difficult to expect, and 90% by weight When added in excess, no marked increase in efficacy effect is seen.

As demonstrated in the examples below, the compositions of the present invention reduce the amount of proinflammatory cytokines such as TNF-α, IL-1β and IL-12 by inducing an immune tolerance response, Increasing the expression levels of antiinflammatory cytokines such as IL-10, Foxp3 and TGF-β increases the Th2 type immune response, which reduces the Th1 type immune response. As a result, joint-specific autoimmune and inflammatory reactions are reduced, thereby exerting or preventing arthritis.

The composition of the present invention exhibits very good efficacy in the prevention or treatment of arthritis (osteoarthritis and rheumatoid arthritis), preferably inflammatory disease rheumatoid arthritis as a kind of autoimmune disease. In addition, since the components used as an active ingredient in the composition of the present invention is already recognized safety, the composition of the present invention is also very safe for the human body. The active ingredients included in the composition of the present invention are excellent safety recognized as food ingredients, it is surprising that these ingredients exhibit superior efficacy to prevent or treat arthritis than conventional medicine (eg, methotrexate).

The limitation of conventional arthritis treatment is that it is difficult to develop arthritis-specific therapeutic agents due to the wide variety of autoantigens involved in the immune response, and the conventional arthritis treatment drugs developed are simply drugs that inhibit only the inflammatory response. There was a problem that several side effects exist. In addition, most of the health foods for the treatment of arthritis had a fragmentary function mainly composed of glucosamine complex and shark cartilage extract, which are one of the cartilage components. In contrast, the composition of the present invention can prevent and treat arthritis by effectively controlling the autoimmune response and inflammation that occur specifically in the joint without side effects by inducing immune tolerance occurring in the intestinal system immune system.

On the other hand, according to a third embodiment of the present invention, the present invention provides a pharmaceutical composition for treating dry eye syndrome comprising a fish eye extract.

The present inventors have tried to develop a composition for treating dry eye syndrome, which has excellent efficacy of treating dry eye syndrome and greatly reduces side effects. As a result, when the fish eye extract is used for dry eye syndrome, the moisturizing effect and corneal epithelial cell disorder were quickly treated, and it was confirmed that dry eye syndrome could be treated.

In the composition of the present invention, the basic active ingredient is a fish eye extract. Fish eye debris in the present invention exhibits an eye moisturizing effect, a healing effect of corneal epithelial cell disorders by itself, but is used as an extract form to extract the active ingredient in order to increase the efficacy of treating and preventing dry eye syndrome without feeling foreign bodies. It is desirable to.

In the present invention, the fish eye extract is used as a polar solvent or a mixed solvent of a lower (C1-C4) alcohol such as water, methanol, or ethanol of 2 to 20 times, preferably about 2 to 5 times, to fish eye shreds. The extract obtained by cold extraction, reflux cooling extraction, hot water extraction, ultrasonic extraction, ultra high pressure extraction, etc. at 10 ° C to 30 ° C extraction temperature is filtered, concentrated under reduced pressure, or lyophilized to obtain an extract available in a polar solvent. It is possible.

In the present invention, when the ultra-high pressure extraction method is used for the extraction, the elution of useful components due to the destruction of fish eye tissue becomes easy, and weak bonds such as hydrogen bonds, electrical bonds, van der Waals bonds, and hydrogen bonds with limited energy levels The binding by these components can be separated and the new material can be eluted, the cytotoxic substances are destroyed, the important components can be extracted within a short time, and the extract has almost no impurities, and it is easy to obtain a single component of high purity. have.

The ultra-high pressure extraction in the present invention is preferably treated for 5 to 60 minutes at a pressure of 100 to 3000MPa, 40 to 90 ℃ temperature, more preferably at a pressure of 500 to 1000MPa, 40 to 60 ℃ temperature for 10 to 30 minutes. When the pressure condition is a low pressure condition of 100MPa or less, or when the extraction time is within 5 minutes, the ocular cell tissue destruction is insufficient, the elution efficiency of useful components is low, the pressure condition is a high pressure condition of 3000MPa or more, or extraction If the time is 60 minutes or more, the energy efficiency required for extraction is reduced, so it is preferable to extract in the above range, and 40 to 90 ℃ temperature conditions to effectively remove weak bonds such as hydrogen bonds, electrical bonds, van der Waals bonds Preference is given to performing at. In addition, the pH at the time of ultra-high pressure extraction is not limited to a specific pH, but preferably it is preferable to lower the pH of the water inside the ultra-high pressure to 1 to 6. When the pH is adjusted within the above range, the dissolution efficiency of the useful ingredient is increased.

In addition, the present invention is subjected to reflux extraction of the ocular extract of the fish subjected to the ultra-high pressure treatment using an extraction solvent selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane, hexane and mixtures thereof. After cooling, the filtered filtrate is more preferably concentrated under reduced pressure and freeze-dried at 50 占 폚 or lower.

The composition for treating dry eye syndrome of the present invention is formulated in the form of eye drops using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. It may be prepared in unit dosage form or by incorporation into a large volume container.

At this time, the fish eye extract may be prepared by including 0.01 to 10% (w / v) based on the total composition, when less than 0.01 can not fully achieve the dry eye treatment effect, 10% (w / v) In the above case, since the increase in the effect of treating dry eye syndrome is insignificant, the above range is preferable.

In the composition for treating dry eye syndrome of the present invention, the viscosity is set according to the kind of pharmacologically active substance, the concentration in the drug, the concentration of the electrolyte in the eye, and the use of the ideal composition and the dosage range. For wound healing by eye surgery or foreign body and alleviation of foreign substance inflammation or irritable immune response, relatively medium to high concentrations of drugs are ideal, and the frequency of administration may vary to some extent depending on the severity of dry eye syndrome being treated. In two to four eye drops per day (eg, 24 hours).

In addition, the composition for treating dry eye syndrome of the present invention may further include one or more selected from the group consisting of anti-inflammatory drugs and steroidal or nonsteroidal anti-inflammatory drugs.

Anti-inflammatory drugs that can be used in the present invention include dexamethasone, fluorometholone, prednisolone, bromfenac, diclofenac, prubipropene, ketorolac, and salts thereof. Preferably, the anti-inflammatory drug is dexamethasone 0.01 to 2.0% (w / v), fluorometholone 0.01 to 2.0% (w / v), prednisolone 0.01 to 5.0% (w / v), bromfenac 0.001 to 1.0% ( w / v), diclofenac 0.05-1.0% (w / v), frubiprofen 0.005-0.5% (w / v), ketorolac 0.005-1.0% (w / v), and salts thereof At least one selected, and more preferably, fluorometholone and diclofenac can be used.

On the other hand, the composition of the present invention may further comprise one or more adjuvants selected from the group consisting of preservatives, isotonic agents, stabilizers, buffers and pH adjusting agents.

The auxiliary agents are those conventionally used in the art to which the present invention pertains, and there are no particular limitations as they vary depending on the respective purposes, and follow the general pH, osmotic pressure and viscosity ranges of the ophthalmic composition. However, in consideration of the effect on the active ingredients, it is preferable to add in an amount of 0.001 to 20% (w / v) based on the total composition, preferably in an amount of 0.01 to 10% (w / v).

In addition, tears are the same as the osmotic pressure of 0.9% (w / v) sodium chloride solution corresponding to physiological saline solution, including polymers, glycoproteins, minerals (vitamine, etc.) and inorganic salts. The ophthalmic composition of the present invention is preferably adjusted to about 290 mOsmol / kg osmotic pressure of physiological saline when administered to the eye, the range is 270 to 310 mOsmol / kg.

Osmotic pressure-controlling material of the composition for treating dry eye syndrome used in the present invention is at least one selected from the group consisting of D-sorbitol, glycerin, concentrated glycerin, mannitol and maltitol syrup and ionic substances, the composition ratio of which is one volume The weight ratio is about 0.5 to 20%, preferably 1 to 10%.

In particular, as a stabilizer added to form a natural polymer into an aqueous eye drop, sodium etate 0.05 to 0.2% (w / v), ε-aminocaproic acid 0.01 to 0.05% (w / v), sodium sulfate anhydrous 0.01 to 0.2 One or more selected from the group consisting of% (w / v), ascorbic acid 0.02 to 0.5% (w / v), citric acid 0.3 to 2.0% (w / v) and the like can be used.

In the present invention, as a preservative, benzalkonium chloride 0.002 to 0.1% (w / v), cerimide 0.002 to 0.01% (w / v), chimerosal 0.001 to 0.01% (w / v), chlorobutanol 0.25 to 0.5% (w / v), polyhexamethylene biguanide 0.002 to 0.1% (w / v), paraoxybenzoic acid alkyl methyl, paraoxybenzoic acid ethyl, paraoxybenzoic acid propyl or paraoxybenzoic acid butyl 0.001 to 0.05% (w / v), At least one selected from the group consisting of dihydroacetic acid 0.001 to 0.1% (w / v), chlorocresol 0.0001 to 0.05% (w / v), and chlorohexidine 0.001 to 0.01% (w / v) can be used. have.

On the other hand, in the path of light transmission to the retina, the cornea, which is a transparent tissue, plays a major role in refraction and transmission of light, and is a tissue in which abundant nerves are distributed instead of blood vessels. The growth of corneal cells is differentiated from basal cells to superficial cells, and in normal eyes, it is known that about 1 to 2 weeks have elapsed until the drop of superficial cells. The incidence distribution of anterior segment trauma is mainly targeted to the cornea and eyelids, and its cause can be divided into chemical damage and physical damage, and physical damage can be divided into external foreign matter and surgical damage. have.

The loss of the corneal epithelium is mainly targeted by the surface and wing cell layers, and the adjacent epithelial cells slide to cover the epithelial defect and to heal by mitosis of the basal cells. Among these healing mechanisms, the inflammatory response is known to be caused by the infiltration of white blood cells into the injury site through the limbus, and the steroid system is prescribed as a drug that suppresses excessive inflammatory response. In general, steroid wound healing eye drops are known to reduce inflammatory cell infiltration and prevent the release of proteolytic enzymes by using them for the first 10 days. Necrosis of the corneal stromal layer is known to promote the breakdown of the collagen fibers and to inhibit the damage site repair process. In particular, long-term administration is known to cause glaucoma, cataracts, bacterial conjunctivitis.

When the fish eye extract of the present invention is used as an ophthalmic composition containing a pharmacologically active substance (active ingredient) for ophthalmic treatment, tissues in the eye, in particular, are formed by forming a gel having good adhesion to the damage site on the corneal conjunctival epithelial cells. It protects the corneal epithelial cell layer and induces growth, and promotes fibrin production of the cells, thereby promoting tissue production of the damaged area of the eye to help repair the damaged area.

Therefore, the ophthalmic composition comprising the fish eye extract of the present invention as an active ingredient has a prophylactic or therapeutic effect against corneal conjunctival epithelial disorders. In particular, it is of particular value as a keratoconjunctival epithelial disorder drug that reduces the side effects of long-term use of existing steroid or nonsteroidal drugs.

According to the present invention can be applied to the skin cosmetics and can provide a crush or extract of fish eye excellent in safety because it is harmless to the human body.

In addition, the present invention can provide a cosmetic composition excellent in the skin whitening effect, skin moisturizing effect, skin elasticity improving effect using a crushed product or extract of fish eye.

In addition, the present invention can provide a cosmetic composition having excellent cell activating effect using the lysate or extract of the fish eye has excellent skin regeneration and wound healing effect, and also has a very good stimulating effect.

In addition, the present invention can provide a composition that can prevent and treat arthritis by effectively controlling the autoimmune response and inflammation that occur specifically in the joint without side effects by inducing immune tolerance that occurs in the intestinal immune system.

In addition, the present invention by providing an ophthalmic composition comprising a fish eye extract, it is possible to manufacture a drug for treating dry eye syndrome with an excellent healing effect on small wounds and hydrophobic surgery, wounds due to chemical damage.

1 is a graph showing the results of analysis of the arthritis prevention efficacy of the composition containing the fish eye extract of the present invention as an active ingredient using AIA (adjuvant induced arthritis) induced rats.

Figure 2 is a graph showing the results of analyzing the efficacy of treating arthritis of the composition containing the fish eye extract of the present invention as an active ingredient using an AIA-induced rat.

Figure 3 shows the results of experiments comparing the efficacy of the arthritis treatment of the composition containing the fish eye extract of the present invention as a composition, the mixed composition of the present invention and a therapeutic agent for arthritis (methotrexate: MTX).

4 is a result of analyzing the effect on the expression of proinflammatory cytokines of the composition containing the fish eye extract of the present invention as an active ingredient by real-time PCR method using mixed lymphocytes.

5 is a result of analyzing the effect on the cell proliferation of the composition containing the fish eye extract of the present invention as an active ingredient using a mixed lymphocyte around the joint.

Figure 6 is a graph showing the results of the analysis of the arthritis prevention efficacy of the composition containing the fish eye fragments of the present invention as an active ingredient using AIA (adjuvant induced arthritis) induced rats.

Figure 7 is a graph showing the results of analyzing the efficacy of treating arthritis of the composition containing the fish eye fragments of the present invention as an active ingredient using an AIA-induced rat.

FIG. 8 shows the results of experiments comparing the efficacy of treating arthritis of a composition containing a fish eye lysate of the present invention with each composition, the mixed composition of the present invention, and an arthritis therapeutic agent (metotrexate: MTX).

9 is a result of analyzing the effect on the expression of proinflammatory cytokines of the composition containing the fish eye lysate of the present invention as an active ingredient by real-time PCR method using mixed lymphocytes.

10 is a result of analyzing the effect on the cell proliferation of the composition containing the fish eye lysate of the present invention as an active ingredient using a mixed lymphocyte around the joint.

Figure 11 is a graph showing the water evaporation of the agar medium over time after the addition of the composition comprising a fish eye extract of the present invention.

12 is a graph showing the damage area ratio over time after inducing corneal epithelial damage of the composition comprising a fish eye extract of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes all modifications of equivalent technical spirit.

실시예 1, 6 : 참다랑어 또는 고등어 안구의 파쇄물 제조Examples 1 and 6: Preparation of Shreds of Bluefin or Mackerel Eyes

Fresh bluefin tuna or tuna was quenched and separated from meat to obtain eyeballs and frozen. Frozen bluefin tuna eyes were homogenized using a homogenizer.

실시예 2, 7 : 참다랑어 또는 고등어 안구의 추출물 제조Example 2, 7: Preparation of extract of bluefin tuna or mackerel eye

After quenching fresh bluefin tuna or mackerel, the frozen eye obtained by separating the meat and extracted with 100% 120 ℃ for about 2 to 3 hours using a 70% ethanol aqueous solution.

실시예 3, 8 : 참다랑어 또는 고등어 안구의 추출물 제조Example 3, 8: Preparation of extract of bluefin tuna or mackerel eye

After cooling the fresh bluefin tuna or mackerel, the frozen eye obtained by separating meat from the meat was heated to 50 ° C. under a pressure of 1000 MPa under a pressure of 1000 MPa, treated for 30 minutes, and cooled to room temperature. The ultrahigh pressure bluefin tuna eye extract was extracted and refluxed three times for 5 hours using purified water, and then filtered using Whatman # 10 filter paper. The filtrate thus obtained was finally filtered using a filter of 0.25 uM. When the filtration was completed, the obtained filtrate was transferred to a concentration tank, concentrated under reduced pressure and freeze-dried at 50 ℃ or less. The final extract was prepared using one or more solvents of purified water, ethanol, butylene glycol, and propylene glycol so that the obtained vacuum concentrate and lyophilisate contained 0.001 to 70.0 wt%.

실시예 4, 9 : 참다랑어 또는 고등어 안구의 추출물 제조Example 4, 9: Preparation of extract of bluefin tuna or mackerel eye

The extract of bluefin tuna eyeballs was prepared in the same manner as in Examples 3 and 8 except that the ultrahigh pressure treated bluefin tuna extract was refluxed with an aqueous 70% (v / v) ethanol solution.

실시예 5, 10 : 참다랑어 또는 고등어 안구의 추출물 제조Example 5, 10: Preparation of extract of bluefin tuna or mackerel eye

The extract of bluefin tuna eyeballs was prepared in the same manner as in Examples 3 and 8, except that the ultrahigh-pressure treated bluefin tuna extract was refluxed with 30% (v / v) butylene glycol.

비교예 1 : 소 안구의 파쇄물의 제조Comparative Example 1 Preparation of Crushed Bovine Ocular

Except for using bovine eye instead of bluefin tuna or mackerel was prepared in the same manner as in Example 1 bovine eye crush.

비교예 2 : 소 안구의 추출물의 제조Comparative Example 2: Preparation of Bovine Eye Extracts

An extract of bovine eyeballs was prepared in the same manner as in Example 2 except that bovine eyeballs were used instead of bluefin tuna or mackerel.

비교예 3 : 소 안구의 추출물의 제조Comparative Example 3: Preparation of Bovine Eye Extracts

Except for using bovine eye instead of bluefin tuna or mackerel was prepared in the same manner as in Example 3 bovine eye crush.

비교예 4 : 소 안구의 추출물의 제조Comparative Example 4: Preparation of Bovine Eye Extracts

Except for using bovine eye instead of bluefin tuna or mackerel was prepared in the same manner as in Example 4 bovine eye crush.

비교예 5 : 소 안구의 추출물의 제조Comparative Example 5: Preparation of Bovine Eye Extracts

Except for using bovine eyeballs instead of bluefin tuna or mackerel was prepared in the same manner as Example 5 bovine eyeballs.

시험예 1 : B16F1 멜라노싸이트를 이용한 멜라닌 생성 억제효과 측정Test Example 1: Determination of melanin production inhibitory effect using B16F1 melanocytes

In order to confirm the whitening effect of the lysate or extract obtained in Examples 1 to 10 and Comparative Examples 1 to 5, the test example measured the degree of inhibition of melanin production against B16F1 melanocytes.

The B16F1 melanocytes used in this test example are cell strains derived from mice, and cells that secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to evaluate the degree of melanin black pigment reduction. The B16F1 melanosite used in this test example was used after being sold from ATCC (American Type Culture Collection, Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed in 6-well plates at a concentration of 2 x 10 ⁶ per well, and cells were attached and then incubated for 72 hours by treating the samples at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, the cell number was measured, and the cells were recovered by centrifugation.

Quantification of intracellular melanin was performed by slightly modifying the method of Rotan ( Cancer Res. , 40: 3345-3350, 1980). The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with a microplate reader, and then quantify the melanin. Melanin production inhibition rate (%) was measured. Melanin inhibition rate (%) of the B16F1 melanocytes was calculated by the equation (1), IC ₅₀ value is the concentration of the substance inhibits 50% melanogenesis.

Equation 1

A: Melanin amount in wells without sample

B: Melanin amount in the wells to which the sample was added

The results of testing the melanin production inhibitory effect of B16F1 melanocytes are shown in Table 1 below.

Table 1

	Melanin production inhibitory effect (IC ₅₀ )
Example 1 (Fragment of Bluefin Tuna Eyeball)	0.30
Example 2 (extract of bluefin tuna eye)	0.31
Example 3 (extract of bluefin tuna eye)	0.38
Example 4 (extract of bluefin tuna eye)	0.25
Example 5 (extract of bluefin tuna eye)	0.32
Example 6 (Crushed Mackerel Eyeball)	0.35
Example 7 (extract of mackerel eye)	0.34
Example 8 (extract of mackerel eye)	0.40
Example 9 (extract of mackerel eye)	0.33
Example 10 (extract of mackerel eye)	0.36
Comparative Example 1 (Crushed Bovine Eye)	0.51
Comparative Example 2 (Extract of Bovine Eye)	0.50
Comparative Example 3 (Extract of Bovine Eye)	0.75
Comparative Example 4 (Extract of Bovine Eye)	0.42
Comparative Example 5 (Extract of Bovine Eye)	0.55
Arbutin	0.33

As shown in Table 1, the crushed or extract of the eye of the bluefin tuna or mackerel showed a significantly higher melanin production inhibitory effect than the crushed or extract of bovine eye, and has a melanin production inhibitory effect equal to or higher than the conventional whitening agent arbutin Indicated.

시험예 2 : Test Example 2: in vitroin vitro MMP-1 inhibition assay MMP-1 inhibition assay

In this test example, the substrate metalloproteinase (MMP-1) inhibitory activity of the lysate or extract of Examples 1 to 10 and Comparative Examples 1 to 5 was measured in a biochemical model, which was purified collagenase and its collagen. It is based on the use of collagen and gelatin conjugated to a substrate, fluorescein (EnzChek ™ Gelatinase / Collagenase Kit, Molecular Probe).

Collagenase purified from Clostridium histocomum was supplied in the EnzChek ™ gelatinase / collagenase kit. A reaction buffer consisting of DQ-collagen purified from porcine skin and conjugated to fluorescein with 0.05M Tris-HCl, 0.15M NaCl, 5mM CaCl2 and 0.2mM sodium azide (pH 7.6) was found in EnzChek® gelatinase / Collagenase kit (Molcula Probes) was used. The lysates or extracts of Examples 1 to 10 and Comparative Examples 1 to 5 were dissolved in the reaction buffer. 0.02; Test at 0.04% (w / v). Dilutions of the test were incubated with 25 ug / ml of DQ-collagen and 0.1 U / ml of collagenase for 15 minutes, 45 minutes, and 120 minutes at room temperature.

In each experimental condition, the control corresponding to the collagenase and DQ-collagen mixture was also incubated in the same manner.

Also in each experimental condition a sample called Blank, hereinafter 'enzyme free blank', was incubated in the presence of DQ-collagen and in the absence of collagenase. Each experiment was performed three times.

After 15, 45 and 120 minutes, the signal corresponding to the degradation of DQ-collagen was measured with a fluorometer (excitation: 485 nm, emission 505 nm).

The fluorescence value of each sample was measured based on the 'enzyme-free blank' fluorescence value. Results are expressed as percent variance for fluorescence units per sample and control.

TABLE 2

	Inhibition Rate (%) Treatment Concentration 0.02%	Inhibition Rate (%) Treatment Concentration 0.04%
Example 1 (Fragment of Bluefin Tuna Eyeball)	50	60
Example 2 (extract of bluefin tuna eye)	53	62
Example 3 (extract of bluefin tuna eye)	42	51
Example 4 (extract of bluefin tuna eye)	60	68
Example 5 (extract of bluefin tuna eye)	54	61
Example 6 (Crushed Mackerel Eyeball)	50	58
Example 7 (extract of mackerel eye)	51	59
Example 8 (extract of mackerel eye)	44	52
Example 9 (extract of mackerel eye)	58	65
Example 10 (extract of mackerel eye)	51	60
Comparative Example 1 (Crushed Bovine Eye)	37	43
Comparative Example 2 (Extract of Bovine Eye)	38	44
Comparative Example 3 (Extract of Bovine Eye)	34	40
Comparative Example 4 (Extract of Bovine Eye)	41	47
Comparative Example 5 (Extract of Bovine Eye)	38	44
Retinol	0	5
Green tea extract	46	55

According to Table 2 above, eye debris or extracts tested at 0.02, 0.04% (v / v) retained anticollagenase / gelatinase activity in a dose dependent manner. In addition, the crush or extract of bluefin tuna or mackerel eyes inhibited about 52% to 68% of the collagen degrading activity of Clostridium collagenase, respectively, when treated with 0.04%, which was much superior to the inhibitory effect of green tea extract.

시험예 3 : 자외선 조사 후 MMP-1 발현억제 평가Test Example 3 Evaluation of MMP-1 Expression Inhibition After UV Irradiation

In this test example, ELISA was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the debris or extracts of Examples 1 to 10 and Comparative Examples 1 to 5.

Human dermal fibroblasts were irradiated with UVA at an energy of 5 J / cm 2 using a UV chamber. UV irradiation dose and culture time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. Negative controls were wrapped in tinfoil and maintained at the same time in the environment of UVA. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact with the previously dispensed medium and irradiated with UVA and then exchanged with the medium containing the sample for 24 hours of incubation, and then the medium was recovered and coated on a 96-well plate. The primary antibody (MMP-1 (Ab-5) monoclonal antibody and MMP-2 (Ab-3) monoclonal antibody) was treated and reacted at 37 ° C. for 60 minutes. After reacting the secondary antibody anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (1mg / mlρ-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes and then the microplate reader was reacted. Absorbance is measured at 405 nm. As a control, one without addition of a sample was used.

TABLE 3

	Treatment concentration (%)	% Inhibition of MMP-1 expression
Example 1 (Fragment of Bluefin Tuna Eyeball)	0.1	20
Example 2 (extract of bluefin tuna eye)	0.1	21
Example 3 (extract of bluefin tuna eye)	0.1	18
Example 4 (extract of bluefin tuna eye)	0.1	24
Example 5 (extract of bluefin tuna eye)	0.1	21
Example 6 (Crushed Mackerel Eyeball)	0.1	20
Example 7 (extract of mackerel eye)	0.1	21
Example 8 (extract of mackerel eye)	0.1	16
Example 9 (extract of mackerel eye)	0.1	23
Example 10 (extract of mackerel eye)	0.1	20
Comparative Example 1 (Crushed Bovine Eye)	0.1	13
Comparative Example 2 (Extract of Bovine Eye)	0.1	14
Comparative Example 3 (Extract of Bovine Eye)	0.1	11
Comparative Example 4 (Extract of Bovine Eye)	0.1	16
Comparative Example 5 (Extract of Bovine Eye)	0.1	14
Retinol	0.1	21
Control	-	-

As shown in Table 3, the crush or extract of bluefin tuna or mackerel eye showed a high inhibition rate compared to the control group without treatment for MMP-1 expression induced during ultraviolet irradiation, which was used as a positive control retinol The result was equal to or higher than the inhibition rate of.

시험예 4 : 자외선조사에 의한 세포독성 완화 효과Test Example 4: Cytotoxic alleviation effect by ultraviolet irradiation

This test example was carried out to evaluate the mitigating effect of cytotoxicity by ultraviolet irradiation of the lysate or extract of Examples 1 to 10 and Comparative Examples 1 to 5.

Fibroblasts (fibroblasts) were placed in a 24-well test plate 1 x 10 ⁵ and attached for 24 hours. Each well was washed once with PBS and 500 ul of PBS was added to each well. The fibroblasts were irradiated with UV 10mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then the PBS was removed and 10% FBS was added to the cell culture medium (DMEM). 1 ml) was added. After treating the ultra-high pressure extract to the eye to be evaluated therein was incubated for 24 hours. After 24 hours, the medium was removed, and 500 μl of cell culture medium and 60 μl of MTT solution (2.5 mg / ml) were added to each well, followed by incubation in a 37 ° C. CO ₂ incubator for 2 hours. The medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added. Shake the cells for 5 minutes to lyse the cells and transfer 100 μl of the supernatant to 96-well test plates and measure the 565 nm absorbance in a microplate reader. Cell survival rate (%) was measured by Equation 2, and cytotoxicity remission rate by UV light was calculated by Equation 3.

Equation 2

Bo: 565 nm absorbance of wells that only reacted with cell culture media

Bt: 565 nm absorbance of the wells that developed the colorless reaction wells

St: 565 nm absorbance of the wells that reacted the sample treated wells

Equation 3

Bo: cell viability of wells without UV irradiation and no sample treatment

Bt: cell viability of wells that were not irradiated with UV light

St: Cell survival rate of wells treated with UV

Table 4

	Treatment concentration (%)	Cytotoxic alleviation rate (%)
Example 1 (Fragment of Bluefin Tuna Eyeball)	0.01	38
Example 1 (Fragment of Bluefin Tuna Eyeball)	0.02	60
Example 2 (extract of bluefin tuna eye)	0.01	39
Example 2 (extract of bluefin tuna eye)	0.02	61
Example 3 (extract of bluefin tuna eye)	0.01	33
Example 3 (extract of bluefin tuna eye)	0.02	54
Example 4 (extract of bluefin tuna eye)	0.01	41
Example 4 (extract of bluefin tuna eye)	0.02	63
Example 5 (extract of bluefin tuna eye)	0.01	38
Example 5 (extract of bluefin tuna eye)	0.02	60
Example 6 (Crushed Mackerel Eyeball)	0.01	37
Example 6 (Crushed Mackerel Eyeball)	0.02	60
Example 7 (extract of mackerel eye)	0.01	37
Example 7 (extract of mackerel eye)	0.02	60
Example 8 (extract of mackerel eye)	0.01	31
Example 8 (extract of mackerel eye)	0.02	50
Example 9 (extract of mackerel eye)	0.01	39
Example 9 (extract of mackerel eye)	0.02	62
Example 10 (extract of mackerel eye)	0.01	36
Example 10 (extract of mackerel eye)	0.02	59
Comparative Example 1 (Crushed Bovine Eye)	0.01	29
Comparative Example 1 (Crushed Bovine Eye)	0.02	45
Comparative Example 2 (Extract of Bovine Eye)	0.01	29
Comparative Example 2 (Extract of Bovine Eye)	0.02	46
Comparative Example 3 (Extract of Bovine Eye)	0.01	22
Comparative Example 3 (Extract of Bovine Eye)	0.02	38
Comparative Example 4 (Extract of Bovine Eye)	0.01	31
Comparative Example 4 (Extract of Bovine Eye)	0.02	47
Comparative Example 5 (Extract of Bovine Eye)	0.01	30
Comparative Example 5 (Extract of Bovine Eye)	0.02	45

As shown in Table 4, the crush or extract of the bluefin tuna or mackerel eye to reduce the cytotoxicity caused by ultraviolet light by about 50 ~ 62% at 0.02% concentration to effectively protect the cytotoxicity caused by ultraviolet light even at low concentrations there was.

시험예 5 : 세포 배양기술을 이용한 자극완화 효과 평가Test Example 5 Evaluation of Stimulation Relaxation Effect Using Cell Culture Technology

This test example is a sodium lauryl sulfate (Sodium Lauryl Sulfate) which is a substance causing skin irritation by using a cell culture technology in order to evaluate the skin irritation effect of the crush or extract of Examples 1 to 10, Comparative Examples 1 to 5 Stimulating effect was evaluated to the extent of inhibiting apoptosis by SLS).

SLS is a substance that is used as a criterion for evaluation of skin irritation based on cosmetics, and is a substance that binds to cell membranes to inhibit cell metabolism and destroys cell membranes to cause skin irritation.

This test is to evaluate the effect of reducing SLS apoptosis when SLS and ultra high pressure ocular extracts are added to human fibroblasts at the same time.

The Hs68 fibroblasts used in this test example were human dermal dermal cells and were used by the ATCC (American Type Culture Collection, Accession No .: 1635).

Stimulation relaxation test using the cell culture technology was carried out as follows.

Human-derived fibroblasts were dispensed at a concentration of 1 X 10 ⁵ per well in a 96-well plate incubator, and cultured for 24 hours, followed by 0.002% SLS alone, 0.002% SLS, and Examples 1 to 10 and Comparative Examples 1 to 5, respectively. The lysate or extract was treated simultaneously with 0.1% and further incubated for 24 hours. After incubation, MTT (Sigma) reagent was added and incubated for 4 hours, the culture medium was removed, 1N NaOH / isopropanol solution was added, stirred for 20 minutes, and the absorbance was measured at 565 nm with an absorbance spectrometer. Table 5 shows the effect of inhibiting apoptosis.

Table 5

Sample name	Fibroblast survival rate (%)
0.002% SLS	41
0.002% SLS + Example 1 (Crushed Bluefin Eyeball)	76
0.002% SLS + Example 2 (extract of bluefin tuna eye)	77
0.002% SLS + Example 3 (extract of bluefin tuna eye)	74
0.002% SLS + Example 4 (extract of bluefin tuna eye)	80
0.002% SLS + Example 5 (extract of bluefin tuna eye)	77
0.002% SLS + Example 6 (Crushed Mackerel Eyeball)	75
0.002% SLS + Example 7 (extract of mackerel eye)	75
0.002% SLS + Example 8 (extract of mackerel eye)	72
0.002% SLS + Example 9 (extract of mackerel eye)	78
0.002% SLS + Example 10 (extract of mackerel eye)	75
0.002% SLS + Comparative Example 1 (Crushed Bovine Ocular)	69
0.002% SLS + Comparative Example 2 (extract of bovine eye)	70
0.002% SLS + Comparative Example 3 (extract of bovine eye)	66
0.002% SLS + Comparative Example 4 (extract of bovine eye)	73
0.002% SLS + Comparative Example 5 (bovine eye extract)	70

As shown in Table 5, the crush or extract of the bluefin tuna or mackerel eye effectively inhibits apoptosis caused by SLS to reduce the skin irritation that can occur as a cosmetic substrate when prescribed with a cosmetic substrate I could see that.

실시예 11, 12 및 비교예 6 : 화장료 제조Examples 11, 12 and Comparative Example 6: Cosmetic Preparation

In the present Example, a cosmetic was prepared using the extracts obtained in Examples 4, 9 and Comparative Example 4. The prepared cosmetics were in the form of a cream, the composition of which is shown in Table 6.

The 'Na' phase recorded in Table 6 was heated and stored at 70 ° C. After preserving emulsification by adding the 'ga' phase to the preserved 'na' phase, the emulsion was uniformly emulsified with a homomixer and then gradually cooled. Creams (Examples 11 and 12 and Comparative Example 6) were prepared.

Table 6

Raw material		Example 11	Example 12	Comparative Example 6	Reference Example
end	Stearyl alcohol		8	8	8	8
	Stearic acid	2	2	2	2
	Stearic Acid Cholesterol	2	2	2	2
	Squalane	4	4	4	4
	2-octyldodecyl alcohol	6	6	6	6
	Polyoxyethylene (25 mol addition) alcohol ester	3	3	3	3
	Glyceryl Monostearic Acid Ester	2	2	2	2
I	Example 4 (extract of bluefin tuna eye)	5	-	-	-
	Example 9 (extract of mackerel eye)	-	5	-	-
	Comparative Example 4 (Extract of Bovine Eye)	-	-	5	-
	Na-hyaluronic acid salt	-	-	-	5
	Propylene glycol	5	5	5	5
	Purified water	Balance against 100	Balance against 100	Balance against 100	Balance against 100

Unit: weight%

시험예 6 : 피부 보습 효과 측정Test Example 6 Measurement of Skin Moisturizing Effect

In this test example, the skin moisturizing effect of the cosmetics prepared in Examples 11 and 12, Comparative Example 6 and Reference Example was evaluated by performing a comparative experiment.

30 subjects (females in their 30s and 40s) were subjected to the cream prepared in Example 11 on the right side of 10 faces, the cream prepared in Comparative Example 6 on the left side of the face, and on the right side of the other 10 faces. Apply the cream prepared in Example 12, the cream prepared in Comparative Example 6 on the left side of the face, the cream prepared in the reference example on the right side of the remaining 10 faces, and the cream prepared in Comparative Example 6 on the left side of the face, respectively. It was applied twice a day for four consecutive weeks. Moisturization of the subject's test site before and after product application (0 week) and 2 and 4 weeks after product application was measured using a Corneometer. There were no adverse events in all subjects who participated in the 4-week trial.

Corneometer measures the amount of water content by measuring the small amount of current conducted to the sensor embedded in the probe head when the probe is contacted with the surface of the skin, and the higher the value, the higher the water content of the skin. .

Table 7 shows the skin moisturizing results of the subjects using the creams prepared in Examples 11 and 12 Comparative Example 6 and Reference Examples.

TABLE 7

Sample name	Product application period
Sample name	0	2	4
Example 11	64.17	73.54	77.65
Example 12	63.94	72.25	76.88
Comparative Example 6	63.96	70.12	73.78
Reference Example	63.84	68.96	72.60

n = 30, p <0.05

Skin moisturizing improvement rate was calculated by the equation (4), the results are shown in Table 8.

Equation 4

A: 0 days moisturizing measurement result

B: Moisture measurement result for 2 or 4 weeks

Table 8

Sample name	Product application period
Sample name	2	4
Example 11	14.60	21.01
Example 12	13.00	20.24
Comparative Example 6	9.63	15.35
Reference Example	8.02	13.72

n = 30, p <0.05

According to Table 8, the extract of the bluefin tuna or mackerel eyeball was found to have an excellent skin moisturizing effect than the extract of bovine eyeball.

시험예 7 : 피부탄력 개선 효과의 평가Test Example 7 Evaluation of Skin Elasticity Improvement Effect

In this test example, the effects of improving skin elasticity in the human body were evaluated for the creams of Examples 11, 12 and Comparative Example 6 as follows.

Thirty test subjects (females in their 30s and 40s) were subjected to the cream prepared in Example 11 on the right side of 15 faces, the cream prepared in Comparative Example 6 on the left side of the face, and on the right side of the remaining 15 faces. The cream prepared in Example 12 was applied to the left side of the face, and the cream prepared in Comparative Example 6 was applied twice a day for two consecutive months.

The skin elasticity improvement effect after the completion of the experiment was measured using a skin elasticity measuring instrument (cutometer SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are described in Table 9 below as the ΔR8 value of the cutometer SEM 575, where the R8 value represents the nature of the viscoelasticity of the skin.

Table 9

Sample name	Skin elasticity effect (ΔR8)
Example 11	0.37
Example 12	0.35
Comparative Example 6	0.23

n = 30, p <0.05

As shown in Table 9, it was found that the extract of bluefin tuna or mackerel eyeball is much better skin elasticity improvement than the extract of bovine eyeball.

시험예 8 : 피부 전층 결손 모델에서의 상처치유 효과Test Example 8 Effect of Wound Healing in a Whole Skin Defect Model

In this test example, the creams of Examples 11, 12 and Comparative Example 6 were evaluated by performing comparative experiments on the wound healing effect in the skin delamination model in the experimental rats.

After each group was divided into groups of five random weights on the day, a full-thickness wound wound model was prepared on the first day of the test. Wound induction was designated

wounds

1 and 2 in order from the top left to the bottom of the test animal, and

wounds

3 and 4 in the order from the top right to the bottom.

A 10-mm long, vertical epidermal defect was created on the rat's back, and the patch was applied to the patch, and the wound contraction rate was performed for 1, 3, 7, 9, and 14 days. .

The groups were separated as in Table 10 and the samples were administered.

Table 10

Sample name	Dosage	Application area by animal
Control	blank patch	full-thickness wound 1
Positive control group (madecassol)	500 mg / cm ²	full-thickness wound 2
Example 11	500 mg / cm ²	full-thickness wound 3
Example 12	500 mg / cm ²	full-thickness wound 4
Comparative Example 6	500 mg / cm ²	full-thickness wound 5

In order to quantify the wound healing effect, the wound shrinkage was obtained by digitally imaging the wound area at 3, 7, 9, 11, and 14 days after 1 day in the autopsy group with an initial wound area of 100. The wound shrinkage was calculated by Equation 5, and the results are shown in Table 12.

Table 11

	1 day	4 days	7 days	9 days	11 days	14 days
Control	1.05	0.77	0.59	0.44	0.21	0.15
Positive control group (madecassol)	1.06	0.90	0.73	0.59	0.33	0.15
Example 11	1.02	0.72	0.42	0.20	0.10	0.03
Example 12	1.02	0.75	0.45	0.23	0.12	0.08
Comparative Example 6	1.05	0.76	0.52	0.36	0.18	0.10

Unit: cm ² , n = 5, p <0.05

Equation 5

A: Result of measurement of wound on one day

B: Wound measurement results on 4, 7, 9, 11, 14 days

Table 12

	4 days	7 days	9 days	11 days	14 days
Control	26.67	43.81	58.10	80.00	90.48
Positive control group (madecassol)	15.09	31.13	44.34	68.87	85.85
Example 11	29.41	58.82	80.39	90.20	97.06
Example 12	26.47	55.88	77.45	88.24	92.16
Comparative Example 6	27.62	50.47	65.71	82.86	90.48

n = 5, p <0.05

As shown in Tables 11 and 12, in measuring the wound shrinkage rate for 14 days after the wound induction, all wounds gradually decreased during the test period (14 days) by window shrinkage, Example 11, The cream containing the extract of 12 bluefin tuna or mackerel eyeballs was found to have an excellent reduction of the window compared to the cream containing the extract of bovine eyeball and the positive control group.

실시예 1' : 어류 안구 추출물 제조Example 1 ′: Preparation of Fish Eye Extract

Fresh bluefin tuna was quenched and separated from meat to prepare fish eye. The prepared fish eye was washed clean with saline solution, and then homogenized using a homogenizer to prepare fish eye debris.

The fish eye debris thus prepared was placed in an ultra-high pressure treatment machine (Ilshin autoclave, Korea), and the temperature was raised to 50 ° C. under a pressure of 1000 MPa, treated for 30 minutes, and cooled to room temperature.

Ultra-high pressure bluefin tuna eye extracts were purified water (Example 1'-1), final 70% (V / V) ethanol aqueous solution (Example 1'-2), final 30% (V / V) butylene glycol Example 1'-3) was added to reflux three times for 5 hours, and the resultant was cooled and then filtered with Whatman No 10 filter paper. The filtrate thus obtained was finally filtered again using a filter of 0.25 uM, and concentrated under reduced pressure and freeze-dried again at 50 ° C. Purified water 100g, ethanol 100g, butylene glycol 100g was added to the decompressed concentrate and freeze-dried 10g prepared as a solvent to prepare a bluefin tuna eye extract.

Mackerel eye extracts (Examples 1'-4 to 1'-6) and bovine eye extracts were prepared using the same method as the method for preparing bluefin tuna eye extract, except that mackerel and bovine eyeballs were used instead of bluefin tuna eyes. Examples 1'-1 to 1'-3) were prepared.

Table 13

	Eye type	Extraction solvent
Example 1'-1	Bluefin tuna	Purified water
Example 1'-2	Bluefin tuna	70% Ethanol
Example 1'-3	Bluefin tuna	30% Butylene Glycol
Example 1'-4	Mackerel	Purified water
Example 1'-5	Mackerel	70% Ethanol
Example 1'-6	Mackerel	30% Butylene Glycol
Comparative Example 1'-1	small	Jeong Se-soo
Comparative Example 1'-2	small	70% Ethanol
Comparative Example 1'-3	small	30% Butylene Glycol

시험예 1' : 본 발명 조성물의 관절염 예방 및 치료 효능 분석Test Example 1 ': Analysis of efficacy of preventing and treating arthritis of the composition of the present invention

The collagen induced arthritis (CIA) animal model is arthritis induced by the collagen type II collagen, which is similar to human rheumatoid arthritis in pathological, clinical and immunological aspects. This animal model shows autoimmune disease by T cells with collagen specific immune responses and antibodies against autologous antigens such as collagen.

Adjuvant induced arthritis (AIA) animal models are arthritis induced by Mycobacterium tuberculosis rather than autoantigens, showing the pathological condition of chronic arthritis. It is not known to act as an exact autoantigen, but it indicates autoimmune diseases such as joint inflammation and tissue destruction by immune-responsive T cells. This animal model is similar to arthritis that occurs naturally in humans.

Because the exact cause of rheumatoid arthritis is not exactly known, experiments with these two animal models can be used to better evaluate efficacy.

시험예 1'-1: 콜라겐 유도 관절염 동물 모델의 제조Test Example 1'-1: Preparation of Collagen-Induced Arthritis Animal Model

Collagen induced arthritis (CIA) animal models were prepared by injecting collagen, which is believed to be the cause of rheumatoid arthritis, as follows.

4 mg of chicken type II collagen (Sigma) was mixed in 1 ml of 100 mM acetic acid and dissolved at 4 ° C. for 1 day. It was mixed and emulsified in 1 ml of Freund's complete adjuvant (DIFCO) containing 4 mg / ml of Mycobacterium tuberculosis, and then 150 µl (300 µg) at the tail of 6 to 8 week old Lewis rats. Was immunized by intradermal injection. On day 7 after the first immunization, 4 mg of collagen type II collagen of chicken was mixed with 1 ml of 100 mM acetic acid, dissolved at 1 ° C. for 1 day, and then emulsified by mixing with 1 ml of Freund's incomplete adjuvant (DIFCO). 150 μl (300 μg) of the emulsion was injected intradermally into the tail to induce a secondary immunization (boosting) reaction. After 1-2 weeks after the second immunization, erythema or swelling, which is a symptom of arthritis, began to appear gradually. .

시험예 1'-2: 아쥬번트 유도 관절염 동물 모델의 제조Test Example 1'-2: Preparation of Adjuvant Induced Arthritis Animal Model

Adjuvant induced arthritis (AIA) animal models are also widely used as collagen-induced arthritis animal models as animal models with pathological characteristics similar to those of human rheumatoid arthritis.

10 mg of Mycobacterium tuberculosis was pulverized in a powder state and mixed in 1 ml of Freund's incomplete adjuvant (DIFCO). Of these, 100 μl (1 mg) were immunized by intradermal injection into the tail region of 6-8 week old Lewis rats. One week after immunization, symptoms of arthritis began to appear gradually.

시험예 1'-3: 본 발명 조성물의 관절염 예방 및 치료 효능 분석Test Example 1'-3: Analysis of efficacy of preventing and treating arthritis of the composition of the present invention

From the day after the induction of the immune response, the test substance was orally administered using a dietary cradle daily. After selecting 10 Lewis rats per group, one group received 25 g (11.5 g / kg) per day of powdered feed and a mixture of examples and comparative examples using an adjuvant induced arthritis animal model, and another group The group used an adjuvant induced arthritis animal model administered only powdered feed as a negative control. The groups were then compared by measuring joint swelling, joint paw swelling, and progression of arthritis through gross findings such as erythema.

In order to confirm the effect of preventing arthritis, an AIA induced immunization reaction was performed two weeks after oral administration of the compositions of Examples and Comparative Examples. The experiment was conducted for a total of 9 weeks. In addition, to confirm the therapeutic effect of arthritis, the composition of Example 1 was orally administered immediately after the AIA induced primary immunization reaction. The experiment was conducted for a total of 9 weeks. Ten rats were used per group.

The swelling degree of the joints and nodes, erythema, etc. were observed for each foot of the rat belonging to each group, and the clinical scores were calculated according to the scoring indexes described in Tables 14 and 15.

Table 14

Table 15

In the clinical score of FIG. 1, which is an experimental result of the prevention of arthritis, the negative control group is an adjuvant-induced arthritis model, and it can be seen that the symptoms of arthritis appear from about two weeks after inducing an immune response. You can see the symptoms weaken. In Examples 1'-1 to 1'-6 group to which the composition of the present invention was administered, it was found that the symptoms were significantly suppressed, unlike the control group, and that the swelling of the foot was much suppressed compared to the negative control group. have. However, this inhibitory effect was remarkably alleviated in Comparative Example 1'-1 to 1'-3 group to which bovine eye extract was administered. Therefore, it was found that the composition comprising the fish eye extract of the present invention exhibits excellent preventive effect against arthritis.

In the clinical score of FIG. 2, the experimental result of the arthritis treatment effect, the control group was an adjuvant-induced arthritis model. It can be seen that arthritis symptom appears from about 2 weeks after inducing an immune response, and gradually increases after about 5 weeks. It can be confirmed that is weakened. In Examples 1'-1 to 1'-6 group to which the composition of the present invention was administered, it was observed that arthritis symptoms were greatly suppressed, and it was also found that the swelling of the feet was also greatly suppressed. However, this inhibitory effect was not clearly observed in the Comparative Example 1'-1 to 1'-3 group administered the bovine eye extract. Therefore, it can be seen that the composition comprising the fish eye extract of the present invention exhibits excellent therapeutic efficacy against arthritis.

시험예 2' : 관절염 치료 효능 비교 실험Test Example 2 ': Comparative Experiment of Arthritis Treatment Efficacy

The composition of Examples 1'-1 and 1'-4, which was found to be excellent in Test Example 1 ', was used to analyze the efficacy of preventing or treating arthritis more precisely.

The experimental method is similar to Test Example 1'-3. Ten rats were used for each group.

The negative control group was a non-administered group, and the MTX administered to the positive control group was a methotrexate treatment group conventionally used as an arthritis treatment agent, and was administered twice a week at about 60 µg per rat.

Each composition was orally administered for two weeks first, and then a primary immune response using collagen was induced. One week later, a second immune response was induced and the clinical score and foot thickness were measured to compare the respective groups. As can be seen in Figures 3 and 4, arthritis symptoms appeared from about 20 days after the CIA-induced immune response, and in the acute phase up to 35 days, the effect of MTX was superior to the composition of the present invention, but chronic phase thereafter rather, the therapeutic and prophylactic efficacy of the compositions of the present invention was better (FIG. 3).

Therefore, it can be seen that the composition of the present invention exhibits the same or better therapeutic efficacy of arthritis than the conventional arthritis therapeutic agent MTX.

시험예 3' : 친염증·항염증 사이토카인 발현에 미치는 영향분석(관절주위 림프구 이용)Test Example 3 ': Analysis of effect on pro-inflammatory and anti-inflammatory cytokine expression (using periarticular lymphocytes)

Arthritis treatment efficacy experiment was conducted as in Test Example 2 '. At 75 days after arthritis-induced immunization, each group was sacrificed to isolate the popliteal lymph node and inguinal lymph node around the joint, and then the tissues were ground and mixed. (mixed lymphocytes).

Mixed lymphocytes were then aliquoted into 24-well plates at 5 × 10 ⁶ cells / well and stimulated for 24 hours by adding 40 μg / well of type II collagen. Cells were then collected and treated with Trizol to yield total RNA. CDNA was synthesized using oligo dT primer and reverse transcriptase (Promega) from 1 ㎍ of RNA of each experimental group. Subsequently, the synthesized cDNA was used as a template, and quantitative real-time PCR was performed using 10 pmol of primers and SYBR premix for each cytokine to analyze and compare cytokine expression levels (FIG. 4). Quantitative real-time PCR was incubated at 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. 30 sec, 62 ° C. 30 sec, and 72 ° C. 30 sec.

The data in FIG. 4 is a relative value when the value of β-actin, which is a housekeeping gene, is quantified, and the value of the non-treated group, which is a control, is 100. It can be seen that the expression levels of proinflammatory cytokines IL-12, TNF-α and IL-1β are low in the administration group of the composition of the present invention. In particular, in the case of IL-12 and IL-1β, the composition administration group of the present invention can be seen that the expression is reduced by 50% or more than the negative control. In addition, the expression levels of Foxp3, IL-10 and TGF-β having anti-inflammatory function, it can be seen that the composition-administered group of the present invention is higher than the control group.

Thus, it can be seen that oral immunotolerance was induced in lymphocytes around the joint by the composition of the present invention. In particular, the expression level of Foxp3, a regulatory T cell marker, was increased, and the expression level of TGF-β, a cytokine having a suppression function, was also greatly increased.

Taken together, the composition of the present invention induces oral immunotolerance in the lymph nodes around the joints that are directly involved in arthritis, which is the amount of anti-inflammatory cytokines such as Foxp3, IL-10 and TGF-β. It can be seen that it increases the level and suppresses the amount of proinflammatory cytokines exogenous such as IL-12, TNF-α and IL-1β.

시험예 4' : 세포 증식 분석Test Example 4 ': cell proliferation assay

Arthritis treatment efficacy experiment was conducted as in Test Example 2 '. 75 days after arthritis-induced immunization, each group was sacrificed to isolate the popliteal lymph node and inguinal lymph node around the joint at the expense of rats with average clinical scores. (mixed lymphocytes). Mixed lymphocytes were then aliquoted into 96-well plates at 2 × 10 ⁵ cells / well and then stimulated for 56 hours by adding 40 μg / well of type II collagen.

Then, 0.5 μCi [ ³ H] thymidine (Perkin Elmer) was added and pulsed for 16 hours to measure the amount of thymidine incorporation (FIG. 5). The data in FIG. 5 is expressed as a relative value when the negative control group is 10. FIG.

As can be seen in Figure 5, in the group administered the composition of the present invention, the lowest thymidine incorporation was shown. This result indicates that the composition of the present invention induces oral immunotolerance in the lymph nodes around the joint directly related to arthritis, and eventually the immune response to collagen is greatly suppressed.

실시예 1'' : 어류 안구 패쇄물 제조Example 1 '' Preparation of Fish Eye Blockades

Fresh bluefin tuna was quenched, separated from meat and clean, and fish eyes prepared. The prepared fish eye was washed clean with saline solution, and then homogenized using a homogenizer to prepare whatman No. Filtration with 10 filter paper. The filtrate thus obtained was subjected to final filtration using a filter of 0.25 uM to finally prepare bluefin tuna eye debris (Example 1 ''-1).

Mackerel eye debris (Example 1 ''-2) and bovine eye debris (Comparative Example 1 '') using the same method as the method for preparing bluefin tuna eye debris, except that mackerel and bovine eyes were used in place of bluefin tuna eye. ) Was prepared.

시험예 1'' : 본 발명 조성물의 관절염 예방 및 치료 효능 분석Test Example 1 '': Analysis of efficacy of preventing and treating arthritis of the composition of the present invention

시험예 1''-1 : 콜라겐 유도 관절염 동물 모델의 제조Test Example 1 ''-1: Preparation of Collagen-Induced Arthritis Animal Model

4 mg of chicken type II collagen (Sigma) was mixed in 1 ml of 100 mM acetic acid and dissolved at 4 ° C. for 1 day. It was mixed with 1 ml of Freund's complete adjuvant (DIFCO) containing 4 mg / ml of Mycobacterium tuberculosis, and then emulsified. Was immunized by intradermal injection. At 7 days after the first immunization, 4 mg of collagen type II collagen of chicken was mixed with 1 ml of 100 mM acetic acid, dissolved at 1 ° C. for 1 day, and then emulsified by mixing with 1 ml of Freund's incomplete adjuvant (DIFCO). 150 μl (300 μg) of the emulsion was injected intradermally into the tail to induce a secondary immunity (boosting) reaction. After 1-2 weeks after the second immunization, erythema or swelling of arthritis began to appear gradually. .

시험예 1''-2 : 어쥬번트 유도 관절염 동물 모델의 제조Test Example 1 ''-2: Preparation of Adjuvant-Induced Arthritis Animal Model

10 mg of Mycobacterium tuberculosis was pulverized with a grinder and mixed in 1 ml of Freund's incomplete adjuvant (DIFCO). Of these, 100 μl (1 mg) were immunized by intradermal injection into the tail region of 6-8 week old Lewis rats. One week after immunization, symptoms of arthritis began to appear gradually.

시험예 1''-3 : 본 발명 조성물의 관절염 예방 및 치료 효능 분석Test Example 1 ''-3: Analysis of efficacy of preventing and treating arthritis of the composition of the present invention

From the day after the induction of the immune response, the test substance was orally administered using a dietary cradle daily. After selecting 10 Lewis rats per group, one group was administered 25 g (11.5 g / kg) per day of powdered feed and a mixture of examples and comparative examples using an adjuvant induced arthritis animal model, and another group The group used an adjuvant induced arthritis animal model administered only powdered feed as a negative control. The groups were then compared by measuring joint swelling, joint paw swelling, and progression of arthritis through gross findings such as erythema.

For each foot of each group of rats, the degree of swelling of joints and nodes, erythema, and the like were observed, and clinical scores were calculated according to the scoring index described in Tables 14 and 15 above.

In the clinical score of FIG. 6, which is an experimental result of the prevention of arthritis, the negative control group is an adjuvant-induced arthritis model, and it can be seen that the symptoms of arthritis appear from about 14 days after inducing an immune response. You can see that the symptoms gradually weaken. In the Examples 1 ''-1 to 1 ''-2 group administered the composition of the present invention, the score was very low, unlike the control group, indicating that the arthritis symptoms were significantly suppressed. It turns out that much is suppressed. However, in Comparative Example 1 '' group administered bovine ocular debris, there was no significant difference from the negative control group, and after 32 days, arthritis symptoms were higher than that of the negative control group. Therefore, it was found that the composition including the fish eye debris of the present invention exhibits excellent preventive effect against arthritis.

Referring to the clinical score of FIG. 7, which is an experimental result of the arthritis treatment effect, the control group is an adjuvant-induced arthritis model, and it can be seen that arthritis symptom appears from about 14 days after inducing an immune response. You can see the symptoms weaken. In the Examples 1 ''-1 to 1 ''-2 group administered with the composition of the present invention, unlike the negative control, the score was very low, and it was observed that arthritis symptoms were suppressed a lot, and the swelling of the foot was also suppressed a lot. I knew it was. However, in Comparative Example 1 '' group administered bovine ocular debris, there was no significant difference from the negative control group, and after 32 days, arthritis symptoms were higher than that of the negative control group. Therefore, it can be seen that the composition comprising the fish eye debris of the present invention exhibits excellent therapeutic efficacy against arthritis.

시험예 2'' : 관절염 치료 효능 비교 실험Test Example 2 '': Comparative Experiment of Arthritis Treatment Efficacy

Using the compositions of Examples 1 ''-1 and 1 ''-2 found to be excellent in Test Example 1 '', arthritis prevention or treatment efficacy was analyzed more precisely.

The experimental method is similar to Test Example 1 ''-3 above. Ten rats were used for each group.

Each composition was orally administered for two weeks first, and then a primary immune response using collagen was induced. One week later, a second immune response was induced and the clinical score and foot thickness were measured to compare the respective groups. As can be seen in Figures 7 and 8, arthritis symptoms appeared after about 2 weeks after the CIA-induced immune response, Example 1 ''-1, 1 ''-2 has a low internal squeeze compared to the negative control group, Comparative Example Arthritis treatment and prevention efficacy was found to be excellent. On the other hand, the effect of MTX was superior to Examples 1 ''-1 and 1 ''-2 in the acute stage up to 5 weeks, but in Examples 1 ''-1 and 1 'rather than in the chronic phase thereafter. '-2 was better, the composition of the present invention was found to be very excellent in the treatment and prevention of chronic arthritis (Fig. 7).

시험예 3'' : 친염증·항염증 사이토카인 발현에 미치는 영향분석(관절주위 림프구 이용)Test Example 3 '': Analysis of effect on pro-inflammatory and anti-inflammatory cytokine expression (using periarticular lymphocytes)

Arthritis treatment efficacy experiment was conducted as in Test Example 2 ''. 75 days after arthritis-induced immunization, each group was sacrificed to isolate the popliteal lymph node and inguinal lymph node around the joint, and then the tissues were ground and mixed lymphocytes. (mixed lymphocytes).

Mixed lymphocytes were then aliquoted into 24-well plates at 5 × 10 ⁶ cells / well and stimulated for 24 hours by adding 40 μg / well of type II collagen. Cells were then collected and treated with Trizol to yield total RNA. CDNA was synthesized using oligo dT primer and reverse transcriptase (Promega) from 1 ㎍ of RNA of each experimental group. Subsequently, the synthesized cDNA was used as a template, and quantitative real-time PCR was performed using 10 pmol of primers and SYBR premix for each cytokine, and cytokine expression levels were analyzed and compared (FIG. 9). Quantitative real-time PCR was incubated at 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. 30 sec, 62 ° C. 30 sec, and 72 ° C. 30 sec.

The data in FIG. 9 is a relative value when the value of -actin, a housekeeping gene, is quantified and the control group is 100. It can be seen that the expression levels of proinflammatory cytokines IL-12, TNF-α and IL-1β are significantly lower in the administration groups of Examples 1 ''-1 and 1 ''-2. In particular, in the case of IL-12 and IL-1β, it can be seen that the Example 1 ''-1, 1 ''-2 administration group reduced expression by 50% or more than the negative control. In addition, the expression levels of Foxp3, IL-10, and TGF-β having anti-inflammatory function showed that Example 1 ''-1, 1 ''-2 administration group was significantly higher.

Thus, it can be seen that oral immunotolerance was induced in lymphocytes around the joint by the composition of the present invention. In particular, the expression level of Foxp3, a regulatory T cell marker, was increased, and the expression level of TGF-, a cytokine having suppression function, was also increased.

Taken together, the composition of the present invention induces oral immunotolerance in lymph nodes around the joint that are directly involved in arthritis, which indicates the expression level of anti-inflammatory cytokines such as Foxp3, IL-10 and TGF-β. It can be seen that it increases the level and suppresses the amount of proinflammatory cytokines exogenous such as IL-12, TNF-α and IL-1β.

시험예 4'' : 세포 증식 분석Test Example 4 '': Cell Proliferation Assay

Arthritis treatment efficacy experiment was conducted as in Test Example 2 ''. 75 days after arthritis-induced immunization, each group was sacrificed to isolate the popliteal lymph node and inguinal lymph node around the joint at the expense of rats with average clinical scores. (mixed lymphocytes). Mixed lymphocytes were then aliquoted into 96-well plates at 2 × 10 ⁵ cells / well and then stimulated for 56 hours by adding 40 μg / well of type II collagen.

Then, 0.5Ci [ ³ H] thymidine (Perkin Elmer) was added and pulsed for 16 hours, and then thymidine incorporation was measured (FIG. 10). The data of FIG. 10 is expressed as a relative value when the negative control group is 10. FIG.

As shown in FIG. 10, the lowest thymidine incorporation was shown in the Examples 1 ″ -1 and 1 ″ -2 administration groups. These results indicate that the composition of the present invention induces oral immunotolerance in the lymph nodes around the joint directly related to arthritis, and eventually the immune response to collagen is greatly suppressed.

실시예 Ⅰ 내지 Ⅵ : 어류 안구 추출물을 포함하는 조성물의 제조Examples I-VI: Preparation of a Composition Comprising a Fish Eye Extract

To 10 g of ultra-high pressure bluefin tuna eye extract, 100 mL of purified water was added, refluxed three times for 5 hours, and after cooling, the resultant was filtered with Whatman No 10 filter paper. The filtrate thus obtained was finally filtered again using a filter of 0.25 uM, concentrated under reduced pressure and lyophilized again at 50 ° C. to prepare a bluefin tuna eye extract, and a mackerel eyeball extract was prepared using the same method as described above. .

In Example I, 0.01 g of benzalkonium chloride and 0.1 g of sodium etate were completely dissolved in 90 mL of sterile purified water, and 0.1 g of bluefin tuna eye extract was sufficiently hydrated. 0.6 g of sodium dihydrogen phosphate and 0.06 g of sodium dihydrogen phosphate were administered to adjust pH and osmotic pressure. Then, 100 mL of sterile purified water and filtered aseptically with a 0.2 ㎛ filter.

Using the same method as in Example I, the compositions of Examples II to VI and Comparative Example I were prepared using the compositions shown in Table 16 below.

Table 16

		Content of each component in 100 mL total composition
		Example I	Example II	Example III	Example IV	Example Ⅴ	Example VI	Comparative Example I
chief ingredient	Bluefin Tuna Eye Extract	0.1g	0.2 g	0.3 g	-	-	-	-
	Mackerel Eye Extract	-	-	-	0.1g	0.2 g	0.3 g	-
	Small eye extract	-	-	-	-	-	-	0.3 g
Preservative	Benzalkonium chloride	0.01 g	0.01 g	0.01 g	0.01 g	0.01 g	0.01 g	0.01 g
Stabilizer	Sodium etherate	0.1g	0.1g	0.1g	0.1g	0.1g	0.1g	0.1g
pH regulator	Sodium hydrogen phosphate	0.6g	0.6g	0.6g	0.6g	0.6g	0.6g	0.6g
pH regulator	Sodium Dihydrogen Phosphate	0.06 g	0.06 g	0.06 g	0.06 g	0.06 g	0.06 g	0.06 g
solvent	Sterilized Purified Water	Remaining amount	Remaining amount	Remaining amount	Remaining amount	Remaining amount	Remaining amount	Remaining amount

시험예 Ⅰ : 보습 효과Test Example I: moisturizing effect

When the compositions prepared in Examples I to VI and Comparative Example I were added dropwise to 1.5% agar plate medium, the reduction of agar weight due to water evaporation was compared. 1.5% agar medium was autoclaved, and then dispensed into a 60 mm diameter glass plate to solidify, followed by dropping 300 uL, the minimum amount that each eye drop composition can be widely distributed in the medium (Masatsugu N. et al. Cornea 12 (5): 433-436, 1993).

Moisture evaporation with time of the 1.5% agar plate medium of the no addition group was used as a control, and the water evaporation amount of the agar medium according to the composition of Examples I to VI and Comparative Example I was compared and shown in Table 17 and FIG. It was. Table 17 shows the relative weight (%) of agar medium over time.

Table 17

As can be seen in Table 17 and FIG. 11, it was found that the evaporation of water was less in the group to which the eye extracts of Examples and Comparative Examples were added compared to the control group to which nothing was added.

In addition, in the group to which the fish eye extracts of Examples I to VI were added, it was found that water evaporation was suppressed as the amount added increased, and the moisturizing effect also increased depending on the amount of fish eye extract added.

Comparing the groups of Comparative Example I, Example III and Example VI, which added the same amount of eye extract, the water evaporated in a nearly similar pattern, and in Examples III and Example VI, which added fish eye extract, Slightly, evaporation of water was suppressed compared to the comparative example administered with bovine eye extract.

시험예 Ⅱ : 각막 상피세포 장애에 대한 상처 치유 효과Test Example II: Wound Healing Effect on Corneal Epithelial Cell Disorder

As a test animal, six males of 2.3-3.0 kg of New Zealand white rabbits who were healthy and had no eye diseases or abnormalities in the eye were selected and tested in a total of 12 eyes. The rabbits used for the experiment were bred in the kennel and used dry feed for water and rabbits. The rabbits were managed according to 'Regulation of Animal Use and Breeding Management Regulations (October 15, 2004, Food and Drug Administration Regulation No. 116)'.

As an experimental group applied to rabbits, the composition prepared according to Example III (0.3% of bluefin tuna eye extract) and Example VI (0.3% of mackerel eye extract) which showed the best experimental results was used, and Comparative Example I as a control. A composition prepared according to (0.3% bovine eye extract) was used. In addition, a group treated with saline (0.9% NaCl) only as a negative control group. The negative control group was set up to measure the natural healing ability of the cornea and did not receive any other drug ('Effect of Topical Na-Hyaluronan on Hemidesmosome Formation in n-Heptanol-Induced Corneal Injury', JH Chung, et al., (1998) Ophthalmic Res. 30, 96-100).

For the wound healing effect experiment, in a breeding environment set at a constant temperature (23 ° C.) and humidity (50%), topical anesthesia with 0.4% benoxine hydrochloride was applied to New Zealand white rabbits purified for 2 weeks. A round filter paper (5.5 mm diameter) sufficiently moistened with n-heptanol was contacted with the central portion of the cornea for 2 minutes to detach the corneal epithelium and cause damage. After washing with 2 mL of saline solution for 2 minutes, the compositions of 0.9% NaCl saline (control), Example III, Example VI, and Comparative Example I, respectively, were instilled 5 minutes after the induction of corneal injury, and thereafter 3 days later. Eye drops (50 μl / once) were applied. One day after the injury, the solution was stained with 1% Rose Bengal stain solution once a day, and then washed with 0.9% sterile saline solution, and the area of corneal epithelial damage was measured with a digital camera. Observation was carried out for 7 days.

The corneal damage ratio was calculated by the ratio of the area value of the portion stained with the dye solution and the total area of the pupil, and the recovery power was expressed as a relative value of the damage area ratio of one day of observation. In addition, the measurement results for the experimental groups of the Examples and Comparative Examples are shown graphically in FIG.

Table 18

As can be seen in Table 18 and Figure 12, immediately after the induction of damage to the eye cornea and conjunctiva by n-heptanol, a similar degree of injury occurred in all individuals of all groups, and then each saline solution, Example And the area of the damaged portion was reduced as the composition of the comparative example was treated.

However, when comparing the wound healing efficacy comparing the area reduction rate of the damaged part, the composition prepared according to the embodiment of the present invention has a superior wound healing effect than the composition comprising saline-treated control and bovine eye extract. Was observed. In particular, as the experimental period was more than 3 days, 4 days or more, the wound healing effect was confirmed to be sustained with excellent performance, and it can be seen that it is possible to effectively treat eye diseases without side effects even during long-term administration.

Claims

Cosmetic composition characterized in that it contains a crushed product or extract of fish eye as an active ingredient.
The method of claim 1,

The fish eye,

Skate, Cod, Mackerel, Dome, Smelt, Salmon, Sea bream, Threadtail bream, Mawfish, Great white shark, Yellowtail, Pollack, Goldenfin, Carp, Crucian carp, Herring, Marlin, Red snapper, Bluefin tuna, Mullet, Swordfish, Squid, Sandfish, Saury , Sunfish, black sea bream, cutlass, mandarin fish, fish, loach, catfish, crab, minty, yellow sea bream, swordfish, bokbok, yellowfin tuna, blue shark, reference machine, prawn, participant, marlin, larva, lizard rockfish, sardine, horse mackerel, zodiac Cosmetics, characterized in that the eyeball of at least one fish selected from the group consisting of mackerel, jade fish, eel, bottlefish, and minfish.
The method of claim 1,

The cosmetic composition, characterized in that containing the crushed or extract of the fish eye containing 0.0001 to 90.0% by weight based on the total weight of the composition.
The method according to any one of claims 1 to 3,

Cosmetic composition, characterized in that for skin whitening.
The method according to any one of claims 1 to 3,

Cosmetic composition for enhancing skin elasticity.
The method according to any one of claims 1 to 3,

Cosmetic composition for moisturizing the skin.
The method according to any one of claims 1 to 3,

Basic cosmetic formulation consisting of lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) and water-in-oil (W / O) type, oil-in-water and water-in-oil makeup base, foundation, skin cover, lipstick, lip Cosmetic composition, characterized in that the formulation of any one selected from gross, face powder, two-way cake, eye shadow, teak color and eyebrow pencil formulations.
Pharmaceutical composition for the prevention and treatment of arthritis, comprising a crush or extract of fish eye as an active ingredient.
The method of claim 8,

The composition is a pharmaceutical composition for the prevention and treatment of arthritis, characterized in that it induces immune tolerance that occurs in the gut-associated immune system (GALT).
The method of claim 8,

The composition is a pharmaceutical composition for preventing and treating arthritis, characterized in that by inhibiting the expression of the cytokines IL-12, TNF-α and IL-1β.
The method of claim 8,

The composition is a pharmaceutical composition for preventing and treating arthritis, characterized in that to promote the expression of the cytokine Foxp3 and TGF-β.
The method of claim 8,

The composition is a pharmaceutical composition for preventing and treating arthritis, characterized in that it further comprises glucosamine, chondroitin or a combination thereof.
The method of claim 8,

The fish is skate, cod, mackerel, dome, smelt, salmon, sheep, sea-tailed sea bream, anglerfish, great white shark, yellowtail, pollock, bigfin, carp, crucian carp, herring, marlin, red snapper, bluefin tuna, mullet, mackerel, waterfish, Sandfish, Saury, Sunfish, Black sea bream, cutlass, mandarin fish, Bess, loach, catfish, crabfish, mintae, yellow sea bream, swordfish, bokbok, yellowfin, bluefin, reference group, prawns, participant, marlin, larvae, zodiac rockfish, sardine, Pharmaceutical composition for the prevention and treatment of arthritis, characterized in that at least one fish selected from the group consisting of horse mackerel, zodiac fish, mackerel, jade fish, eel, bottlefish, and fish.
The method of claim 8,

The fish eye extract,

Pharmaceutical composition for the prevention and treatment of arthritis, characterized in that extracted by treatment for 5 to 60 minutes at a pressure of 100 ~ 3000MPa, temperature 40 ~ 90 ℃.
The method of claim 8,

The fish eye debris,

A pharmaceutical composition for preventing or treating arthritis, characterized in that the filtrate is filtered after crushing the eyeball of the fish with a mixer or homogenizer.
Food composition for arthritis improvement comprising a lysate or extract of fish eye as an active ingredient.
The method of claim 15,

The composition is a food composition for improving arthritis, characterized in that it induces immune tolerance that occurs in the gut associated lymphatic system (GALT).
The method of claim 16,

The fish is skate, cod, mackerel, dome, smelt, salmon, sheep, sea-tailed sea bream, anglerfish, great white shark, yellowtail, pollock, bigfin, carp, crucian carp, herring, marlin, red snapper, bluefin tuna, mullet, mackerel, waterfish, Sandfish, Saury, Sunfish, Black sea bream, cutlass, mandarin fish, Bess, loach, catfish, crabfish, mintae, yellow sea bream, swordfish, bokbok, yellowfin, bluefin, reference group, prawns, participant, marlin, larvae, zodiac rockfish, sardine, The horse mackerel, zodiac, mackerel, jade fish, eel, bottle, and at least one fish selected from the group consisting of minfish, food composition for arthritis improvement.
The method of claim 16,

The fish eye extract,

Food composition for improving arthritis, characterized in that extracted by treatment for 5 to 60 minutes at a pressure 100 ~ 3000MPa, temperature 40 ~ 90 ℃.
The method of claim 16,

The fish eye debris,

Food composition for improving arthritis, characterized in that the filtrate filtered by crushing the eyeball of the fish with a mixer or homogenizer.
Dry eye disease treatment composition comprising fish eye extract as an active ingredient.
The method of claim 21,

The fish eye extract is a composition for treating dry eye syndrome, characterized in that 0.01 to 10% (w / v) based on the total composition.
The method of claim 21,

A composition for treating dry eye syndrome, characterized in that it further comprises one or more adjuvants selected from the group consisting of preservatives, isotonic agents, stabilizers, antioxidants, and pH adjusting agents.
The method of claim 21,

The fish is skate, cod, mackerel, dome, smelt, salmon, sheep, sea-tailed sea bream, eel, great white shark, yellowtail, pollock, bigfin, carp, crucian carp, herring, marlin, red snapper, bluefin tuna, mullet, mackerel, waterfish, Sandfish, Saury, Sunfish, Black sea bream, cutlass, mandarin fish, Bess, loach, catfish, crabfish, mintae, yellow sea bream, swordfish, bokbok, yellowfin, bluefin, reference group, prawns, participant, marlin, larvae, zodiac rockfish, sardine, Composition for treating dry eye syndrome, characterized in that at least one fish selected from the group consisting of horse mackerel, zodiac fish, mackerel, jade fish, eel, bottlefish, and minfish.
The method of claim 21,

The fish eye extract is a composition for treating dry eye syndrome, characterized in that extracted for 5 to 60 minutes at a pressure 100 ~ 3000MPa, temperature 40 ~ 90 ℃.