KR20240159178A - Anti-inflamatory composition for treating or preventing neurodegenerative disease comprising a hydrolysate of male pupa - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition or an anti-inflammatory pharmaceutical composition. The composition comprises, as an active ingredient, a hydrolysate of a pupa, specifically, a hydrolysate obtained by hydrolyzing a pupa with a proteolytic enzyme. The hydrolysate of a pupa may be hydrolysed by treating it with at least one selected from the group consisting of alcalase, neutrase, and protamax, specifically, a mixture of neutrase and protamax.
The anti-inflammatory composition of the present invention, unlike existing anti-inflammatory substances, is made from the pupa of a safe natural product that can be used for food, and thus has no side effects and excellent safety. In addition, it has been confirmed that the composition has an anti-inflammatory effect because it can effectively inhibit the expression of COX-2 and inhibit NO production.

Description

{ANTI-INFLAMATORY COMPOSITION FOR TREATING OR PREVENTING NEURODEGENERATIVE DISEASE COMPRISING A HYDROLYSATE OF MALE PUPA}

The present invention relates to a composition or anti-inflammatory agent having an anti-inflammatory effect, specifically, an anti-inflammatory composition containing a natural product-derived ingredient as an active ingredient.

Inflammation is a local immune response that occurs when cells or tissues are damaged or destroyed by various factors such as harmful substances or organisms from the outside to minimize the damage and restore the damaged area to its original state. It is a useful defense mechanism for protecting the body and removing products created by tissue damage. As a result of the defense mechanism, pain, swelling, redness, or fever may occur, causing functional disorders. The various factors that cause the inflammation include physical factors such as trauma, burns, frostbite, and radiation; chemical factors such as acid; and immunological factors such as antibody reactions. In addition, it can be caused by blood vessels or hormonal imbalances.

In normal cases, the above inflammation neutralizes or removes the pathogenic factor through an inflammatory response in the body and regenerates damaged tissue to restore normal structure and function. However, if the degree of inflammation exceeds a certain level or becomes chronic and progresses to a disease state such as chronic inflammation, it becomes a problem. In addition to being able to observe inflammatory responses in almost all clinical diseases, it is known that enzymes related to inflammatory responses play an important role in the process of carcinogenesis.

According to recent findings, the progression of inflammatory responses in the body is known to be related to the activity of the COX (cyclooxygenase) enzyme. The COX enzyme is a main enzyme involved in the biosynthesis of prostaglandins present in the body (Smith et al., J. Biol. Chem., 271, 33157 (1996)), and it is known that there are two types of isoform enzymes, COX-1 and COX-2. The COX-1 is constantly present in tissues such as the stomach and kidneys and is involved in maintaining normal homeostasis, whereas the COX-2 is an enzyme that is temporarily and rapidly expressed in cells by mitogens or cytokines during inflammation or other immune responses.

Nitric oxide (NO), another potent inflammatory mediator, is produced from L-arginine by NO synthase (NOS), and is produced in many types of cells by external stresses such as UV, or by substances such as endotoxins or cytokines. Such inflammatory stimuli can induce an inflammatory response by increasing the expression of inducible NOS (iNOS) within cells, thereby inducing NO production within cells, and activating macrophages.

Therefore, in order to effectively relieve inflammation, research is being conducted on substances that can suppress the production of NO. However, some side effects are problematic in the case of anti-inflammatory substances developed through such research. For example, non-steroidal anti-inflammatory drugs used to treat acute or chronic inflammatory diseases such as rheumatoid arthritis are known to cause side effects such as gastrointestinal disorders by inhibiting not only the COX-2 enzyme but also the COX-1 enzyme.

Meanwhile, cosmetics used in daily life are products used to protect the skin and beautify and cleanse it, but when looking at the cosmetic composition, ingredients different from the purpose of skin protection are used as essential for product formation. For example, surfactants, preservatives, fragrances, UV blockers, pigments, and various other ingredients used to provide efficacy or effects can be mentioned. The ingredients essential for the manufacture of the above cosmetics are generally known to cause various side effects such as inflammation, pimples, or swelling on the skin.

In addition, it is a well-known fact that sebum components, sweat components, and fatty acids, high-grade alcohols, and proteins in cosmetics discharged from the body can cause skin inflammation when they are broken down into highly toxic substances by skin-resident bacteria, and that skin inflammation can also be caused by ultraviolet rays from the sun.

As such, cosmetics are always potential causes of adverse skin reactions, and various studies have been conducted to resolve these issues. Currently, non-steroidal substances used for the purpose of relieving irritation and inflammation, such as erythema and edema, include flufenamic acid, ibuprofen, benzydamine, and indomethacin; steroidal substances include prednisolone and dexamethasone; and allantoin, azulene, ε-aminokyproic acid, hydrocortisone, licorice acid, and its derivatives (β-glycyrrhetinic acid, glycyrrhetinic acid derivatives) are known to have anti-inflammatory effects.

However, the indomethacin, which is generally used as an anti-inflammatory agent, is a substance that cannot be used in cosmetics, the amount of hydrocortisone used is limited, and licorice acid and its derivatives are difficult to stabilize or have poor solubility, making it difficult to achieve actual effects due to concentration limitations in actual application. Thus, most of the anti-inflammatory agents known to date have problems in terms of skin safety or stability when compounding cosmetics, and thus their use is limited.

Therefore, there is a need for the development of a substance derived from natural substances that can effectively inhibit the COX-2 enzyme and inhibit the production of NO, while having little or no risk of side effects or cytotoxicity, and thus has an excellent anti-inflammatory effect, thereby suppressing inflammation without side effects and can be used in cosmetics without restrictions on its content.

In particular, research and development on cosmetics using natural raw materials is being actively conducted recently to meet the needs of consumers.

The background technology described above is technical information that the inventor possessed for deriving the present invention or acquired in the process of deriving the present invention, and cannot necessarily be considered as publicly known technology disclosed to the general public prior to the application for the present invention.

10-1403396B 10-1702621B 10-2171133B 10-2353372B 1020170120269A

The purpose of the present invention is to solve the problems of the prior art as described above, and the purpose of the present invention is to provide an anti-inflammatory composition containing a natural product-derived substance as an active ingredient, which is less likely to cause problems related to side effects.

In addition, unlike existing anti-inflammatory substances, it is derived from a natural product, specifically, a pupa of a wasp used as a food, and thus has excellent safety as it is non-cytotoxic, and it has been confirmed that it can effectively inhibit the expression of COX-2 in relation to inflammation and inhibit NO production, and thus the purpose is to provide an anti-inflammatory composition or cosmetic composition for treating, improving or preventing inflammatory diseases.

To achieve the above purpose, the present invention provides an anti-inflammatory composition containing a natural product-derived ingredient, specifically, a hydrolyzed product of a water pupa, as an effective ingredient.

Additionally, the anti-inflammatory composition may be a composition for treating, preventing, or improving an inflammatory disease.

The above hydrolysate of the water pupa may be obtained by hydrolyzing the water pupa with a protein hydrolyzing enzyme.

The above drone pupa may be a drone pupa of the Western honeybee (Apis mellifera).

The above protein hydrolyzing enzyme may be at least one selected from the group consisting of alcalase, neutrase, and protamax, and preferably may be a mixture of neutrase and protamax.

More specifically, the hydrolyzed drone pupa may be obtained by hydrolyzing Western honeybee (Apis mellifera) drone pupa by treating it with neutrase and protamax at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit.

In another aspect, the present invention provides an anti-inflammatory pharmaceutical composition comprising a hydrolysate of a pupa hydrolyzed with a proteolytic enzyme as an effective ingredient.

The above drone pupa may be a drone pupa of the western honeybee ( Apis mellifera ).

The above proteolytic enzyme may be at least one selected from the group consisting of alcalase, neutrase, and protamax.

More specifically, the hydrolyzed drone pupa may be obtained by hydrolyzing Western honeybee (Apis mellifera) drone pupa by treating it with neutrase and protamax at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit.

The above anti-inflammatory pharmaceutical composition may be formulated with at least one selected from the group consisting of ointments, creams, gels, lotions, solutions, dressings, patches, blisters, tapes, mists, external powders, and sprays.

In another aspect, the present invention provides an anti-inflammatory cosmetic composition to achieve the above purpose.

The above drone pupa may be a drone pupa of the western honeybee ( Apis mellifera ).

The above proteolytic enzyme may be at least one selected from the group consisting of alcalase, neutrase, and protamax.

More specifically, the hydrolyzed drone pupa may be obtained by hydrolyzing Western honeybee (Apis mellifera) drone pupa by treating it with neutrase and protamax at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit.

The above anti-inflammatory cosmetic composition may have any one formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a lotion, a powder, a soap, a cleansing oil containing a surfactant, a powder foundation, an emulsion foundation, a wax foundation, a shampoo, a rinse, a tonic, and a spray.

As used herein, singular expressions include plural expressions unless the context clearly indicates otherwise.

Unless otherwise specified herein, terms such as “include” or “have” are intended to specify the presence of a feature, number, step, or combination thereof described in the specification, but should be understood not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, or combinations thereof.

Unless otherwise specified herein, an active ingredient means an ingredient that provides a specific effect in a composition, specifically an anti-inflammatory composition or an anti-inflammatory pharmaceutical composition.

Unless otherwise specified herein, the term "active ingredient" means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself, and specifically means an ingredient that provides an anti-inflammatory effect or an effect of treating, improving, or preventing an inflammatory disease in a pharmaceutical composition, food composition, or cosmetic composition.

Unless otherwise specified herein, a hydrolysate is a substance produced after protein decomposition, also called a protein hydrolysate, and a hydrolysate of a water pupa means a substance produced by hydrolyzing a water pupa protein by a protein hydrolase. The protein hydrolase means an enzyme that performs a chemical reaction by hydrolyzing peptide bonds, etc. in a protein molecule to turn the protein into an amino acid or peptide mixture.

Unless otherwise specified herein, a pharmaceutically or food-wise acceptable carrier means a carrier, excipient or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered hydrolysate.

In this specification, treatment means any act of improving or beneficially changing the symptoms of the disease by administering a composition containing an effective ingredient according to the present invention.

In this specification, prevention means any act of inhibiting or delaying the onset of the disease by administering a composition containing an effective ingredient according to the present invention.

In this specification, an inflammatory disease is defined as any condition that is characterized by a local or systemic biological defense response to external physical or chemical stimuli or infection by external infectious agents such as bacteria, fungi, viruses, and various allergens. This response involves a series of complex physiological reactions such as activation of various inflammatory mediators and enzymes related to immune cells (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-α, IL-6, IL-1β, PGE2), infiltration of body fluids, cell migration, and tissue destruction, and is externally manifested by symptoms such as erythema, pain, edema, fever, and deterioration or loss of specific body functions. Since the above inflammatory disease may be acute, chronic, ulcerative, allergic, or necrotic, as long as any disease is included in the above definition of an inflammatory disease, it does not matter whether it is acute, chronic, ulcerative, allergic, or necrotic.

Specifically, the inflammatory diseases may include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraines, headache, back pain, fibromyalgia, myofascial diseases, viral infections (e.g., hepatitis C), bacterial infections, fungal infections, burns, wounds resulting from surgical or dental work, prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, and the like.

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

The present invention relates to an anti-inflammatory composition comprising a hydrolyzed product of a water pupa as an effective ingredient.

The inventors of the present invention, while studying various materials for the development of an anti-inflammatory composition targeting safe materials with no problematic side effects, selected male pupa that can be used for food and confirmed the antioxidant and anti-inflammatory effects of a hydrolysate obtained by hydrolyzing the male pupa with a proteolytic enzyme. As a result of confirming the anti-inflammatory effect, it was confirmed that a hydrolysate hydrolyzed with a combination of specific proteolytic enzymes has a significantly superior anti-inflammatory effect compared to other hydrolysates, and thus completed the present invention.

The present invention relates to an anti-inflammatory composition comprising a hydrolyzed product of a water pupa as an effective ingredient.

The above male pupa refers to a male pupa, and may be a male pupa of 15 to 25 days old, preferably 16 to 20 days old.

The above-mentioned drone pupae contain various nutrients such as protein, vitamins B, D, E, dietary fiber, and folic acid, which are twice as much as the silkworm pupae used for food, and depending on the life cycle, the composition of vitamins, amino acid composition, fatty acid content, etc. may be different from that of the drone larvae and adults, and thus may exhibit different physiological activities.

The above drone pupae may include all of the native honeybees (oriental species), apiculture honeybees (western species), bumblebees, wasps, hornets, queen bees, unidentified bees, etc., and may include all bees belonging to the taxonomic families of Apidae, Deciduous Wasp Family, Cicada Family, Wasp Family, and Bee Superfamily, but preferably, it may be a drone pupae of the western honeybee ( Apis mellifera ).

The hydrolysate of the above-mentioned drone pupa may be manufactured according to a conventional manufacturing method for manufacturing a hydrolysate of a terrestrial animal, such as drones or drone pupae, and may preferably be manufactured by hydrolysis using an enzyme. More preferably, the hydrolysate of the present invention comprises a drone pupa manufacturing step of washing drone pupae that have been thawed after rapid freezing in running water, drying them with hot air, and pulverizing them to manufacture a drone pupa pulverized product; a substrate solution manufacturing step of mixing a buffer solution with the drone pupa pulverized product to manufacture a substrate solution of 1 to 10% (w/v), preferably 3 to 5% (w/v); an enzyme inactivation step of heating the substrate solution to inactivate an autoenzyme included in the substrate solution; a hydrolysis step of treating the substrate solution with a protein hydrolytic enzyme to make 0.1 to 1% (w/w) of the substrate solution to manufacture a hydrolysate; It may be manufactured by a method for manufacturing a hydrolysate of a water pupa, including a step of manufacturing a supernatant by heating the manufactured hydrolysate to inactivate a protein hydrolysing enzyme, centrifuging the same, and isolating the supernatant; and a step of manufacturing a hydrolysate of a water pupa by filtering the supernatant to obtain a hydrolysate of a water pupa.

In the above hydrolysis step, the protein hydrolyzing enzyme may be at least one selected from the group consisting of alcalase, neutrase, and protamax, preferably a mixture of neutrase and protamax, and more preferably a mixture in which the treatment amounts of neutrase and protamax are mixed at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit.

More specifically, the hydrolyzed drone pupa may be obtained by hydrolyzing Western honeybee (Apis mellifera) drone pupa by treating it with neutrase and protamax at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit.

According to one embodiment of the present invention, it was confirmed that the hydrolysate of the water pupa can effectively inhibit the expression of NOS (iNOS) and inhibit the COX-2 enzyme in relation to the secretion of NO that can directly induce inflammation.

Therefore, an anti-inflammatory composition containing the hydrolyzed product of the above-mentioned water pupa as an effective ingredient can be used to suppress inflammation or to treat, improve or prevent inflammation or inflammatory diseases.

The above inflammation is a concept including general inflammatory diseases, and as an example, the inflammatory disease may be at least one selected from the group consisting of various dermatitis, allergy, systemic lupus erythematosus, retinitis, gastritis, hepatitis, enteritis, pancreatitis, arthritis, and nephritis, and the above allergy includes anaphylaxis, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergy, food allergy, and drug allergy.

The above anti-inflammatory composition can be used to treat, improve or prevent inflammation by effectively inhibiting the expression of NOS (iNOS) that causes inflammation and inhibiting COX-2 enzyme.

Therefore, the anti-inflammatory composition of the present invention can be applied as an anti-inflammatory pharmaceutical composition or a pharmaceutical composition for treating or preventing an inflammatory disease, or as a food composition for preventing or improving the inflammatory disease. The food composition can be, for example, a health functional food composition for preventing or improving the inflammatory disease.

In this aspect, the anti-inflammatory pharmaceutical composition may contain a hydrolyzed product of the pupa as an effective ingredient.

The anti-inflammatory pharmaceutical composition containing the above-mentioned hydrolyzed water pupa as an effective ingredient can be directly applied to animals including humans. The above-mentioned animals are a group of organisms corresponding to plants that mainly consume organic matter as nutrients and have differentiated digestive organs, excretory organs, and respiratory organs, and are preferably mammals, and more preferably humans.

The above-mentioned hydrolysate of the water pupa can be used alone in the above-mentioned anti-inflammatory composition, and may additionally contain a pharmacologically acceptable carrier, excipient, diluent, or auxiliary ingredient. More specifically, when a composition containing the above-mentioned hydrolysate of the water pupa is used as a medicine, or for medicinal or pharmaceutical purposes, the above-mentioned hydrolysate of the water pupa can be used by mixing it with a pharmaceutically acceptable carrier or excipient or by diluting it with a diluent according to a conventional method.

In this case, the content of the hydrolyzed water pupa in the composition may be 0.001 wt% to 99.9 wt%, 0.1 wt% to 99 wt%, or 1 wt% to 50 wt%, but is not limited thereto, and the content of the compound may be appropriately adjusted to a desired content and used depending on the usage aspect and usage method of the composition.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include, but are not limited to, one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin and saline, and any conventional carrier, excipient or diluent can be used. In addition, the pharmaceutical composition may further comprise conventional fillers, bulking agents, binders, disintegrants, anticoagulants, lubricants, wetting agents, pH regulators, nutrients, vitamins, electrolytes, alginic acid and salts thereof, pectic acid and salts thereof, protective colostrum, glycerin, flavoring, emulsifiers, or preservatives. The above components may be added independently or in combination to the active ingredient, the hydrolyzed water pupa.

In addition, the composition of the present invention may further include, in addition to the above-mentioned effective ingredient, a substance recognized as having a known anti-inflammatory effect, for example, a substance used as a COX-2 inhibitor, an NO inhibitor, or an anti-inflammatory agent.

When the above composition is used as a medicine, the administration method can be either oral or parenteral, and for example, it can be administered through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular.

In addition, the formulation of the composition may vary depending on the method of use, and may be formulated using a method well known in the art to which the present invention pertains so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In general, solid preparations for oral administration include tablets, pills, soft or hard capsules, pills, powders and granules, and these preparations may be prepared by mixing one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

Forms for parenteral administration may be creams, lotions, ointments, pastes, liquids and solutions, aerosols, fluid extracts, elixirs, infusions, sachets, patches or injections.

Furthermore, the compositions of the present invention may be formulated using any suitable method known in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The dosage of the above composition can be determined by considering the administration method, the age and sex of the recipient, the severity and condition of the patient, the absorbability of the active ingredient in the body, the inactivation rate, and the concomitantly administered drug, and for example, based on the daily effective ingredient, it can be administered at 0.1 mg/kg (body weight) to 500 mg/kg (body weight), 0.1 mg/kg (body weight) to 400 mg/kg (body weight), or 1 mg/kg (body weight) to 300 mg/kg (body weight), and can be administered once or in several divided doses. The above dosage does not limit the scope of the present invention in any way.

In another aspect, the health functional food composition for preventing or improving the inflammatory disease may contain a hydrolyzed product of the water pupa as an effective ingredient.

A health functional food composition containing the above-mentioned hydrolyzed product of the pupae as an effective ingredient for preventing or improving inflammatory diseases can be directly applied to animals, including humans.

The above health functional food refers to a food group or food composition that has been processed to sufficiently express the body's regulatory functions, such as regulating the biological defense rhythm, preventing disease, and recovering, by using physical, biochemical, or bioengineering techniques to provide added value to the food so that the food's functions can be performed and expressed for a specific purpose. The above health functional food may include food additives that are acceptable in terms of food science, and may further include appropriate carriers, excipients, and diluents that are commonly used in the manufacture of health functional foods.

The health functional food composition for preventing or improving an inflammatory disease of the present invention may contain the hydrolyzed water pupa in an amount of 0.001 wt% to 99.9 wt%, or 0.01 wt% to 50 wt%, or 0.1 wt% to 30 wt%, or 0.1 wt% to 15 wt% of the total food weight.

The present invention also provides a cosmetic composition comprising the hydrolyzed product of the above-mentioned water pupa as an effective ingredient.

The above-mentioned hydrolysate of the water pupa is not only non-cytotoxic because it is derived from a natural substance, but also has an excellent anti-inflammatory effect, so it has excellent anti-inflammatory and anti-irritant activities, and thus can effectively control inflammation induced by ingredients included in cosmetics and inflammation induced by the external environment. Therefore, the above-mentioned hydrolysate of the water pupa can be used as an effective ingredient of a cosmetic composition having an anti-inflammatory improvement and alleviation effect.

The above cosmetic composition can be provided for use as a base cosmetic, a makeup cosmetic, a body cosmetic, a hair cosmetic, a scalp cosmetic, a shaving cosmetic, or an oral cosmetic.

Examples of the above-mentioned basic cosmetics include creams, toners, packs, massage creams, milky lotions, etc., examples of the above-mentioned makeup cosmetics include foundations, makeup bases, lipsticks, eye shadows, eyeliners, mascara, eyebrow pencils, etc., examples of the above-mentioned body cosmetics include soaps, liquid cleansers, bath agents, sunscreen creams, sun oils, etc., examples of the above-mentioned hair cosmetics include shampoos, rinses, hair treatments, hair mousses, hair liquids, pomades, hair colorants, hair bleaches, or color rinses, examples of the above-mentioned scalp cosmetics include hair tonics or scalp treatments, examples of the above-mentioned shaving cosmetics include aftershave lotions or shaving creams, and examples of the above-mentioned oral cosmetics include toothpastes or mouthwashes.

In addition to the effective ingredient, the cosmetic composition may additionally contain ingredients commonly incorporated in cosmetic compositions, such as moisturizers, ultraviolet absorbers, vitamins, plant and animal extracts, digestives, whitening agents, vasodilators, astringents, refreshing agents, and hormones, depending on the intended use and the properties of the cosmetic composition. In addition, the cosmetic composition may additionally contain a mechanism necessary for the penetration or transfer of a drug or effective ingredient into skin tissue.

The formulation of the above cosmetic composition may take an appropriate form depending on the intended use and the properties of the cosmetic composition, and may be, for example, an aqueous solution, a solubilized system, an emulsified system, a liquid system, a gel system, a paste system, an ointment system, an aerosol system, a two-layer water-oil system, or a three-layer water-oil-powder system. The examples of the formulations are merely examples, and the formulation and form of the cosmetic composition of the present invention are not limited by the examples.

The above-mentioned effective ingredient, hydrolyzed water pupa, may be included in an amount of 0.001 wt% to 50 wt% based on the total weight of the above-mentioned cosmetic composition, and preferably 0.01 wt% to 20 wt%, but the content may be appropriately adjusted depending on the content of components other than the effective ingredient contained in the formulation or cosmetic composition, and the content of the effective ingredient included in the present invention is not limited by the above-mentioned content.

The present invention also provides a method for producing the hydrolyzed product of the above-mentioned water pupa.

Specifically, the method for producing hydrolysate of the above-mentioned water pupa may include a step of producing a ground water pupa; a step of preparing a substrate solution; an enzyme inactivation step; a hydrolysis step; a step of preparing a supernatant; and a step of producing a hydrolysate of the water pupa.

More specifically, the method for producing a hydrolysate of a drooping pupa comprises: a drooping pupa producing step of washing drooping pupa, which is thawed after rapid freezing, in running water, and then drying and crushing the drooping pupa to produce a drooping pupa producing step; a substrate solution producing step of mixing a buffer solution with the drooping pupa producing step of producing a substrate solution of 1 to 10% (w/v), preferably 3 to 5% (w/v); an enzyme inactivation step of heating the substrate solution to inactivate an autoenzyme included in the substrate solution; a hydrolysis step of treating the substrate solution with a proteolytic enzyme to an amount of 0.1 to 1% (w/w) of the substrate solution to produce a hydrolysate; a supernatant producing step of heating the produced hydrolysate to inactivate the proteolytic enzyme, centrifuging the same, and separating the supernatant to obtain a supernatant. It may include a step of producing a hydrolyzed water pupa by filtering the supernatant to obtain a hydrolyzed water pupa.

In the above hydrolysis step, the protein hydrolyzing enzyme may be at least one selected from the group consisting of alcalase, neutrase, and protamax, preferably a mixture of neutrase and protamax, and more preferably a mixture in which the treatment amounts of neutrase and protamax are mixed at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit.

Hereinafter, the present invention will be described in more detail through manufacturing examples and experimental examples. These examples are only intended to explain the present invention more specifically, and it will be obvious to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention.

The hydrolysate of the present invention is a natural substance derived from a substance that can suppress NO secretion by inhibiting the expression of inducible NOS (iNOS) within cells related to inflammation, and can effectively inhibit inflammation by inhibiting COX-2 related to prostaglandin biosynthesis. Therefore, the anti-inflammatory composition containing the hydrolysate of the present invention as an effective ingredient can be applied to an anti-inflammatory agent or an anti-inflammatory pharmaceutical composition for treating or preventing diseases related to inflammation by inhibiting inflammation, and an anti-inflammatory cosmetic composition having the characteristic of effectively suppressing skin inflammation caused by an external environment, etc., related to a specific formulation. Therefore, the present invention can be widely used in the medical industry related to the treatment of diseases related to inflammation, and the cosmetic industry related to the control of skin inflammation.

Figure 1 shows the effect of enzymatic hydrolysate of dried pupa on iNOS expression in LPS-induced RAW 264.7 cells in one embodiment of the present invention. Figure 1b shows the results of Western blot analysis to confirm the expression of iNOS, and Figure 1a is a graph showing the average values (n = 3, p < 0.05) of the results of the Western blot analysis for the dried pupa treatment group (Unhydrolyzed), the enzymatic hydrolysate treatment group (NP, 2.1, 20), and the untreated pupa group (Control).
Figure 2 shows the results of one embodiment of the present invention when the enzymatic hydrolysate of dried pupa affects COX-2 expression in LPS-induced RAW 264.7 cells. Figure 2b shows the results of Western blot analysis to confirm COX-2 expression, and Figure 2a is a graph showing the average value (n = 3, p < 0.05) of COX-2 expression in the Western blot analysis results relative to the untreated pupa group (Control) (100%) in the dried pupa treatment group (Unhydrolyzed) and the enzymatic hydrolysate treatment group (NP, 2.1, 20).

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Manufacturing example. Manufacturing of hydrolyzed water pupa

The western honeybee ( Apis mellifera L.) drone pupae used in the examples of the present invention were drone pupae between 16 and 20 days old from western honeybee colonies being raised in apiaries in Gwangju Metropolitan City and Gyeongsangnam-do. The collected drone pupae were rapidly frozen using a rapid freezer (DF3503S, Ilshin Bio Co., Ltd., Republic of Korea) and stored at -20℃ until the experiment.

The above frozen pupae were quickly thawed in running tap water immediately after taking them out of the freezer. To remove residue and foreign substances, the thawed pupae were placed on a sieve, washed once more in running tap water, and finally washed with distilled water. The washed pupae were dried with paper towels. The pupae from which moisture had been removed were dried with hot air at 70℃ for 8 hours using a hot air dryer (P-IOV864, Labmate, Korea), and then ground for 10 minutes in a grinder (HMF-3100S, Hanil Electric Co. Ltd., Korea).

The analytical reagents and organic solvents used in the experiment using the above-mentioned crushed pupa were purchased from Sigma-Aldrich Co. (USA).

10 mM sodium phosphate buffer (pH 8) was added to the above hot-air-dried male pupa hot-air-dried powder to prepare a 4% (w/v, male pupa hot-air-dried powder / sodium phosphate buffer) substrate solution, and the substrate solution was treated using a constant temperature water bath at 90°C for 20 minutes to inactivate the autoenzyme.

Three types of food protein hydrolyzing enzymes, alcalase (Alc), protamax (Pro), and neutrase (Neu), and their mixtures were used. The mixtures were a mixture of alcalase and protamax (alcase+protamax, Alc+Pro, AP), a mixture of alcase and neutrase (alcase+neutrase, Alc +Neu, AN), and a mixture of neutrase and protamax (neutrase+protamax, Neu+Pro, NP). The mixing ratios of the mixtures were 1:0, 1:1, 1:2, and 2:1, respectively, based on the enzyme activity unit (AU), and a total of 12 treatment groups were composed.

The enzyme or the enzyme mixture was added to the substrate solution in which the above-mentioned autoenzyme was inactivated so as to be 0.5% (w/w) of the substrate solution, and hydrolysis was performed for 0 to 30 minutes under the conditions of 55°C and 150 rpm, and when the hydrolysis was completed at each time point, the treatment enzyme was inactivated by heating at 90°C for 10 minutes.

After cooling to 4℃, centrifugation was performed at 4,000 Х g for 15 minutes, and the supernatant protein layer was transferred to a new tube, filtered using 0.45 μm cellulose acetate syringe filters (FJ25ASCCA00AFL01, GVS Life Sciences, USA), and the filtrate was obtained as the final sample, which was stored at -20℃ until use in the experiment.

Example 1. Confirmation of the antioxidant effect of hydrolyzed water pupa

In order to confirm the antioxidant effect of the hydrolysate of the water pupa manufactured in the above manufacturing example, DPPH radical scavenging activity and ABTS radical scavenging activity were confirmed.

First, to confirm DPPH radical scavenging activity, the method of Blios (1958) was modified and performed.

Specifically, 100 μl of a 0.4 mM DPPH solution dissolved in ethanol (EtOH) and 100 μl of a solution of the hydrolysate of the female pupa prepared in the above manufacturing example dissolved in 10 mM sodium phosphate buffer (pH 8) at a concentration of 1 μg/mL were mixed in a 96-well plate and reacted in a darkroom for 30 minutes, and the absorbance was measured at 520 nm using a Microplate Reader (Multiskan GO, Thermo Scientific, Finland). The DPPH radical scavenging activity was calculated using the following calculation formula 1. To confirm the DPPH radical scavenging activity, 10 μg/mL of L-ascorbic acid (aa) was used as a positive control. The calculation results are shown in Table 1 below.

[Calculation formula 1]

Calculation formula = ((Control - Blank) - (Sample O.D - Blank))/(Control - Blank)*100

As shown in Table 1 above, among the hydrolysates hydrolyzed using a mixture of Alcase and Neutrase in a 1:0 ratio based on the enzyme activity unit (alcase + neutrase, Alc + Neu, AN), the treatment groups treated for 15 and 20 minutes, and among the hydrolysates hydrolyzed using a mixture of Neutrase and Protamax in a 2:1 ratio based on the enzyme activity unit (neutrase + protamax, Neu + Pro, NP), the treatment group treated for 20 minutes had a DPPH activity of 60 or higher, which was approximately twice as high as that of the control group, ascorbic acid (10 ug).

In addition, to confirm ABTS radical scavenging activity, the method of Miller et al. and Dudonne et al. was modified and performed.

Specifically, 7 mM ABTS and 2.45 mM potassium persulfate (1/1, v/v) were reacted at room temperature for 16 hours. The ABTS produced by the reaction was diluted with ethanol until the absorbance at 734 nm became 0.7 ± 0.02, and 20 μl of a solution in which the hydrolysate of the female pupa prepared in the above manufacturing example was dissolved in 10 mM sodium phosphate buffer (pH 8) at a concentration of 1 μg/mL and 180 μl of the ABTS solution were added to a 96-well plate, mixed homogeneously for 10 to 20 seconds, left at room temperature for 5 minutes, and then the absorbance was measured at 734 nm using a Microplate Reader (Multiskan GO, Thermo Scientific, Finland). The ABTS radical scavenging activity was calculated using the following calculation formula 2 based on the measurement results. To confirm ABTS radical scavenging activity, 10 ㎍/mL of L-ascorbic acid (aa) was used as a positive control. The calculation results are shown in Table 2 below.

[Calculation formula 2]

Calculation formula = ((Control - Blank) - (Sample O.D - Blank))/(Control - Blank)*100

As shown in Table 2 above, among the hydrolysates hydrolyzed using a mixture of Nutrase and Protamax in a 2:1 ratio based on enzyme activity units, the ABTS radical scavenging activity of the treatment groups treated for 20 and 30 minutes was confirmed to be over 70, which was approximately twice as high as that of the control group, ascorbic acid (10 ug), and was confirmed to have the highest antioxidant activity among the 12 hydrolysates.

Based on the above experimental results, the anti-inflammatory effect of the hydrolysate hydrolyzed using a mixture of Nutrase and Protamax, which were confirmed to have the best antioxidant activity, in a 2:1 ratio based on enzyme activity units was confirmed.

Example 2. Confirmation of the inhibitory effect of hydrolyzed water pupa on iNOS and COX-2 protein expression

In order to investigate the anti-inflammatory effect of hydrolysate of dried pollack, which was hydrolyzed using a mixture of Nutrase and Protamax in a ratio of 2:1 based on enzyme activity units, which was confirmed to have the best antioxidant activity in Example 1, on the inhibition of protein expression of iNOS and COX-2 in mouse-derived macrophage RAW264.7 cells (KCLB NO. 40071, Korean Cell Line Bank), an experiment was conducted using the method of the literature (Kim et al ., European Journal of Pharmacology, 721, pp. 267 - 276 (2013)), and the results are shown in Figs. 1 and 2.

Specifically, RAW264.7 cells, which are mouse-derived macrophages of Example 1, were seeded at a concentration of 3 X 10 ⁶ cells/ml in a 60 mm dish and cultured for 12 hours. Then, the hydrolysate of the dried pupa isolated in Example 1 was added at a concentration of 20 μg/ml and pretreated for 3 hours, followed by treatment with 1 μg/ml of lipopolysaccharide (LPS) and cultured for 24 hours to induce an inflammatory response, and then cultured for 24 hours in an incubator at 37°C and 5% carbon dioxide. After the culture, lysis buffer (RIPA (89900, Thermo)) was added and reacted at 4°C for 30 minutes, followed by centrifugation at 4°C and 12,000 rpm for 20 minutes to separate the supernatant. Protein quantification in the separated supernatant was performed using a BSA protein assay kit (Pierce Biotechnology).

iNOS, COX-2, and GAPDH antibodies used in this experiment were purchased from Cell signaling technology (USA) and Santa Cruz (USA). Peroxidase-labeled donkey anti-rabbit and peroxidase-labeled sheep anti-mouse immunoglobulin used as secondary antibodies for the following Western blot analysis were purchased from Sigma-aldrich (USA).

Thereafter, the expression of anti-inflammatory proteins was confirmed through western blot analysis. Specifically, the separated supernatant was electrophoresed for 2 hours using 7.5% or 12% SDS-polyacrylamide gel, and then transferred from the polyacrylamide gel using a nitrocellulose membrane (NC membrane). The transferred nitrocellulose membrane was stopped for 1 hour in fresh blocking buffer (blocking buffer, 0.1% Tween 20 in Tris-buffered saline) containing 5% skim-milk. After the reaction was stopped, the membrane was washed three times with PBST (PBS, 0.1% Tween 20) once every 10 minutes, and then antibodies (Ab) of iNOS (SC-650, Santacruz biotechnology, USA) and COX-2 (SC-1745, Santacruz biotechnology, USA) were diluted 1:1000 and added, and the reaction was performed for 1 hour. After the reaction, the membrane was washed three times with PBST once every 10 minutes, and the secondary antibody (Anti-rabbit IgG, SC-2004, Santacruz biotechnology, USA) was diluted 1:1000 and added, and the reaction was performed for 1 hour.

After the reaction, the mixture was washed three times with PBST once every 10 minutes, and ECL (Amersham Pharmacia Biotech) solution was mixed well in a 1:1 ratio and poured onto a nitrocellulose membrane (NC membrane), and then exposed to X-ray film in a darkroom and developed. The content of actin was measured using an antibody for actin (SC-1616, Santacruz biotechnology, USA) in the same manner.

As a result of the above measurement, the iNOS protein expression level inhibitory effect (Figs. 1a and 1b) and COX-2 protein expression level inhibitory effect (Figs. 2a and 2b) of the hydrolyzed water pupa were specifically confirmed, and this is shown in Figs. 1 and 2.

As shown in the above Figure 1, when hydrolyzed water pupa was added, iNOS expression was significantly reduced in LPS-induced RAW 264.7 cells compared to when dried water pupa was added (Figure 1a), and COX-2 expression was also significantly reduced (Figure 2a).

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims (10)

An anti-inflammatory composition comprising hydrolysate of water parsley as an effective ingredient. In the first paragraph,
The above hydrolyzed water pupa is an anti-inflammatory composition characterized in that the water pupa is hydrolyzed with a protein hydrolyzing enzyme. In the first paragraph,
An anti-inflammatory composition characterized in that the above-mentioned drone pupa is a drone pupa of a western honeybee ( Apis mellifera ). In the second paragraph,
An anti-inflammatory composition, characterized in that the proteolytic enzyme is at least one selected from the group consisting of alcalase, neutrase, and protamax. In paragraph 4,
An anti-inflammatory composition characterized in that the above proteolytic enzyme is a mixture of neutrase and protamax. In the first paragraph,
The above hydrolyzed male pupa is an anti-inflammatory composition obtained by hydrolyzing male pupa of Western honeybees ( Apis mellifera ) by treating them with neutrase and protamax at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit. An anti-inflammatory pharmaceutical composition comprising, as an active ingredient, a hydrolysate of a water pupa hydrolyzed with a proteolytic enzyme. In Article 7,
An anti-inflammatory pharmaceutical composition characterized in that the above-mentioned drone pupa is a drone pupa of a western honeybee ( Apis mellifera ). In Article 7,
An anti-inflammatory pharmaceutical composition, characterized in that the proteolytic enzyme is at least one selected from the group consisting of alcalase, neutrase, and protamax. In Article 7,
The above hydrolysate of male pupa is an anti-inflammatory pharmaceutical composition obtained by hydrolyzing male pupa of Western honeybees ( Apis mellifera ) by treating them with neutrase and protamax at a ratio of 2:1 (neutrase: protamax) based on the enzyme activity unit.