KR102152077B1 - Papain enzyme treated silkworm segment extracts having excellent skin anti-inflammatory effect and use thereof - Google Patents

Abstract

The present invention relates to a papain enzyme decomposition product derived from a cocoon with excellent skin inflammation alleviation activity and a use thereof. Specifically, the present invention relates to a method for producing a papain enzyme decomposition product of a cocoon having an activity to inhibit or alleviate skin inflammation, and a cosmetic composition for alleviating skin inflammation, a health functional food, and a pharmaceutical composition for preventing or treating skin inflammation, which comprise a papain enzyme decomposition product for an ultrasonic extract of cocoon prepared by the above method as an active ingredient. The papain enzyme decomposition product of the cocoon according to the present invention has an excellent inhibitory activity of COX-2 and PGE2, which are factors causing skin inflammation, and at the same time can inhibit phosphorylation of extracellular-signal-regulated kinase (ERK). Therefore, the product can be usefully used in the manufacture of cosmetics, medicines, and health functional foods for the prevention, alleviation, or treatment of skin inflammation.

Description

Papain enzyme treated silkworm segment extracts having excellent skin anti-inflammatory effect and use thereof {Papain enzyme treated silkworm segment extracts having excellent skin anti-inflammatory effect and use thereof}

The present invention relates to a cocoon-derived papain enzyme decomposition product having excellent skin inflammation improving activity, a method for preparing the same, and a use thereof.

The skin is a part of the body that is directly exposed to the external environment, and when exposed to excessive ultraviolet rays or pollutants, skin irritation such as erythema, swelling, and itching and inflammatory reactions are induced. Skin troubles caused by these stressors are not only a cosmetic problem, but it is also known that substances generated in the inflammatory reaction process incidentally cause pigmentation of the skin and accelerate the breakdown of skin elastic fibers, thereby affecting the increase of skin wrinkles. It is covered.

The inflammatory response in the skin begins as an action to defend against skin damage caused by physical stimulation, chemical substances, bacteria, etc., and various immune cells and inflammation-inducing cytokines are involved. TNF-α (tumor mecrosis factor-α), IL-1β (interleukin-1β), IL-6 (interleukin-6), IL-8 (interleukin-8), etc. are typical inflammation-inducing cytokines. In addition, activated macrophages further activate the inflammatory process by excessively producing nitric oxide (NO) or prostaglandin E2 (PGE2), as well as inflammation-inducing cytokines.

In addition, the inflammatory response of the skin is regulated by a number of complex genes, and the major genes are cyclooxygenase-2 (COX-2) and inducible nitric oxide synthases (iNOS). Among them, COX-2 is an inflammation-inducing cytokine, It has been shown to cause PGE2 production and inflammatory skin diseases such as psoriasis, ringworm, and lupus erythematosus by cell growth factor, cancer cell promoting factor, and reactive oxygen species (ROS).

Therefore, substances capable of inhibiting the expression or activity of such skin inflammation inducing factors can be used as skin inflammation improvement and therapeutic agents.

On the other hand, most of the currently developed skin inflammation improvement or treatments are mainly synthetic compounds, and these have a problem that causes side effects in the body and the effect of improving skin inflammation is insufficient. There is a need to develop a therapeutic agent.

Republic of Korea Patent Publication 10-2012-0118188

Accordingly, an object of the present invention is to provide a method for preparing a papain enzyme decomposition product of a cocoon having an activity to inhibit or improve skin inflammation.

Another object of the present invention is to provide a cosmetic composition for improving skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

Another object of the present invention is to provide a health functional food for preventing or improving skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

In order to achieve the object of the present invention as described above, the present invention is to obtain an ultrasonic extract by adding sodium hydroxide solution to a cocoon and ultrasonicating it; And it provides a method for producing a papain enzyme decomposition product of a cocoon having an activity to inhibit or improve skin inflammation, comprising the step of hydrolyzing by adding a papain enzyme to the ultrasonic extract.

In one embodiment of the present invention, the sodium hydroxide solution may be a sodium hydroxide solution having a concentration of 0.1 to 1N.

In one embodiment of the present invention, the ultrasonic treatment may be performed at a temperature of 24 to 26°C at 20 to 30 kHz for 2 to 6 hours.

In addition, the present invention provides a cosmetic composition for improving skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

In one embodiment of the present invention, the papain enzyme decomposition product for the ultrasonic extract of the cocoon is added to the cocoon with a sodium hydroxide solution having a concentration of 0.1 to 1N, and then 24 to 30 kHz at 20 to 30 kHz for 2 to 6 hours. After sonication at a temperature of 26°C to obtain an ultrasonic extract, papain enzyme may be added to the ultrasonic extract, followed by hydrolysis.

In one embodiment of the present invention, the papain enzyme digestion product for the ultrasonic extract of the cocoon is COX-2 (Cyclooxygenase-2) inhibition, PGE2 (Prostaglandin E2) inhibition or ERK (extracellular-signal-regulated kinase) It may be to inhibit the phosphorylation of.

In addition, the present invention provides a health functional food for preventing or improving skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

In one embodiment of the present invention, the papain enzyme decomposition product for the ultrasonic extract of the cocoon is added to the cocoon with a sodium hydroxide solution having a concentration of 0.1 to 1N, and then 24 to 30 kHz at 20 to 30 kHz for 2 to 6 hours. After sonication at a temperature of 26°C to obtain an ultrasonic extract, papain enzyme may be added to the ultrasonic extract, followed by hydrolysis.

In one embodiment of the present invention, the papain enzyme digestion product for the ultrasonic extract of the cocoon is COX-2 (Cyclooxygenase-2) inhibition, PGE2 (Prostaglandin E2) inhibition or ERK (extracellular-signal-regulated kinase) It may be to inhibit the phosphorylation of.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

In one embodiment of the present invention, the papain enzyme decomposition product for the ultrasonic extract of the cocoon is added to the cocoon with a sodium hydroxide solution having a concentration of 0.1 to 1N, and then 24 to 30 kHz at 20 to 30 kHz for 2 to 6 hours. After sonication at a temperature of 26°C to obtain an ultrasonic extract, papain enzyme may be added to the ultrasonic extract, followed by hydrolysis.

In one embodiment of the present invention, the papain enzyme digestion product for the ultrasonic extract of the cocoon is COX-2 (Cyclooxygenase-2) inhibition, PGE2 (Prostaglandin E2) inhibition or ERK (extracellular-signal-regulated kinase) It may be to inhibit the phosphorylation of.

In one embodiment of the present invention, the skin inflammation may be selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea, and acne.

The present invention has established a method for producing a papain enzyme digestion product of a cocoon having an activity of inhibiting or improving skin inflammation, and the papain enzyme digestion product for the ultrasonic extract of a cocoon prepared by the method of the present invention is a skin inflammation Cosmetics, pharmaceuticals, and health functional foods for the prevention, improvement or treatment of skin inflammation as it has excellent inhibitory activity of COX-2 and PGE2, which are inducers, and at the same time inhibits phosphorylation of ERK (extracellular-signal-regulated kinase). It can be usefully used in the manufacture of.

1 shows the result of measuring the concentration of protein extracted from cocoon by reaction time extracted using sodium hydroxide solution by concentration.
Figure 2 shows the results of measuring the concentration of protein extracted from the cocoon according to the ultrasonic treatment time in sodium hydroxide solution of 0.1N and 0.25N concentration.
3 shows the result of measuring the change in molecular weight according to the enzyme reaction time after adding papain enzyme to the cocoon extract extracted by sonication in a 0.1N sodium hydroxide solution.
Figure 4 shows the effect of inhibiting the expression of COX-2 and inhibiting the production of PGE2 on the hydrolyzed products of cocoon extracts extracted by ultrasonic treatment in sodium hydroxide solution of 0.1N concentration using different types of proteolytic enzymes. will be.
FIG. 5 shows the results of confirming the ERK phosphorylation inhibitory effect of papain enzyme digestion for cocoon extract extracted by ultrasonic treatment in sodium hydroxide solution of 0.1N concentration through Western blot.

The present invention is characterized by providing a method for preparing a papain enzyme decomposition product derived from cocoon having excellent skin inflammation improving activity.

Cocoon contains a large amount of proteins such as Serisin and Fibroin, and development is actively underway to use these useful proteins as materials for functional food and beverage.

However, in the case of silk proteins collected as cocoons, when used without undergoing any special treatment process, or when using a purified aqueous fibroin solution by dialysis treatment, the structural characteristics of the fibers and the amino acid sequence of the silk protein It has problems such as deterioration of its value as a functional material, such as decomposition in the human body is delayed or the absorption rate is lowered due to the like.

Therefore, the present inventors are conducting research to obtain a material having excellent skin inflammation improvement or alleviation activity by using a cocoon containing a large amount of useful silk protein in the body, and the ultrasonic extract obtained by ultrasonic treatment on the cocoon was used as a protein. It was confirmed that the hydrolyzate hydrolyzed by using a degrading enzyme has excellent skin inflammation improvement activity.

Accordingly, the present invention can provide a method for preparing a papain enzyme decomposition product of a cocoon having skin inflammation improving activity, and preferably, the method is to obtain an ultrasonic extract by adding sodium hydroxide solution to the cocoon and ultrasonicating it. Step to do; And hydrolyzing by adding papain enzyme to the ultrasonic extract.

More specifically, the method is described above, first by ultrasonic treatment on the cocoon to obtain an ultrasonic extract.

Conventionally, an extraction method using an organic solvent is used to obtain silk protein from cocoon, but there is a problem that the activity of the extracted silk protein is reduced or deformation is caused due to the use of a high concentration of organic solvent, so a low concentration of organic solvent is used. Technologies that can be used are under study.

While the present inventors are also conducting research to obtain silk protein derived from cocoon that has excellent skin inflammation improving activity while using a low concentration of organic solvent, when ultrasound is performed in a low concentration of organic solvent, the silk protein is extracted in high yield. It was confirmed that it can be done.

Therefore, sodium hydroxide solution may be used as the organic solvent that can be used in the present invention, preferably a solution having a concentration of 0.1 to 1N may be used, more preferably a solution having a concentration of 0.1 to 0.3N may be used, Even more preferably, a sodium hydroxide solution having a concentration of 0.1N may be used.

In addition, the ultrasonic treatment can be performed at a temperature of 24 to 26°C at 20 to 30 kHz for 2 to 6 hours under sodium hydroxide solution containing cocoon, and preferably, ultrasonication at 25°C at a frequency of 20 Hz for 4 hours. Treatment was carried out.

When the ultrasonic treatment conditions are performed under the above range, there may be a problem that the silk proteins extracted from the cocoon are decomposed, lose their activity, or denatured due to too high energy, whereas the ultrasonic treatment conditions are less than the above range. If it is carried out as, there is a problem that the extraction yield of silk protein from the cocoon is insufficient. Therefore, it is preferable to perform the ultrasonic treatment within the range established in the present invention.

After obtaining the ultrasonic extract from the cocoon, it is hydrolyzed by adding a proteolytic enzyme to the ultrasonic extract.

At this time, the present inventors obtained hydrolysates using various types of proteolytic enzymes to obtain silk peptides derived from cocoons with excellent skin inflammation improving activity, and analyzed their activity. As a result, papain enzymes were used. It was found that the obtained enzymatic decomposition product was significantly superior in skin inflammation improvement activity compared to the group treated with other enzymes, namely bromelain, pancreatin and proteases.

Therefore, through these results, the present inventors first identified that in order to obtain a silk peptide having excellent skin inflammation improving activity from a cocoon, papain enzyme should be used as a proteolytic enzyme.

In addition, in the present invention, when papain is used as a proteolytic enzyme in the ultrasonic extract, the papain may be used in an amount of 0.5 to 1.0% based on the total weight of the ultrasonic extract.

In one embodiment of the present invention, in order to analyze whether papain enzyme digestion product (hydrolyzate) is active in improving or alleviating skin inflammation with respect to the ultrasonic extract of cocoon, the ability to inhibit COX-2 expression was measured, bromelain, pancreatin Alternatively, it was found that papain hydrolyzate had the best COX-2 expression inhibitory ability compared to the hydrolyzate treated with the enzyme of proteinase. In addition, it was found that the papain hydrolyzate of the present invention was the most excellent in inhibiting PGE2 activity.

In addition, in another embodiment of the present invention, as a result of measuring the phosphorylation of ERK to analyze the effect on the activation of ERK, which is a major factor in the cell signaling process involved in the inflammatory response, bromelain, It was found that papain hydrolyzate inhibited the phosphorylation of ERK most effectively compared to the hydrolyzate treated with pancreatin or the enzyme of proteinase.

On the other hand, various mediators, such as cytokines and prostaglandin E2 (PGE2), are involved in the skin inflammatory response, especially cytokines, tumor necrosis factor-α, TNF-α, and lipopoly. Stimulation such as saccharide (lipopolysaccharide, LPS) moves into the nucleus of a cell and activates NF-κB (Nuclear factor-κB) and AP-1 (activating protein 1). NF-κB and AP-1 are transcription factors that play an important role in the expression of genes that regulate various cellular activities such as cell differentiation, inflammatory response, and cell adhesion, and their activation is cyclooxygena, an enzyme important in inflammation. Aze-2 (cyclooxygenase-2, COX-2) is overexpressed, resulting in the production of excess PGE2, causing inflammation. In addition, when the signaling system of ERK is activated by phosphorylation of ERK, the expression and activity of various inflammatory factors are promoted to induce inflammation.

Therefore, a substance capable of inhibiting COX-2 and PGE2 and inhibiting the activity of ERK can be used as a material capable of preventing and treating skin inflammation.

In this respect, the papain enzyme decomposition product for the ultrasonic extract of the cocoon according to the present invention can inhibit the expression of COX-2 and PGE2, which are skin inflammation inducing factors, and at the same time inhibit the phosphorylation of ERK, thereby preventing skin inflammation, It can be used in the manufacture of cosmetics, health functional foods and pharmaceuticals for improvement or treatment.

Therefore, the present invention can provide a cosmetic composition for improving skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

In the present invention, the cosmetic composition is a softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, It may be a formulation selected from the group consisting of body oil, body essence, makeup base, foundation, hair dye, shampoo, rinse and body cleanser.

The cosmetic composition of the present invention may be prepared in various forms according to a conventional cosmetic preparation method using a papain enzyme decomposition product for the ultrasonic extract of the cocoon, and a stabilizer commonly used in the cosmetic composition field, Conventional adjuvants such as solubilizing agents, vitamins, pigments, and perfumes may be included.

In addition, when the cosmetic composition is manufactured in the form of a fragrance product, shampoo, hair lotion, hair cream, hair gel, etc. containing a papain enzyme decomposition product for the ultrasonic extract of the cocoon, a conventional cleansing solution, astringent solution And it can be used after being diluted with a moisturizing solution.

The cosmetic composition of the present invention is particularly preferably prepared in the form of a lotion, lotion, cream, or essence.

In the cosmetic composition of the present invention, the papain enzyme decomposition product for the ultrasonic extract of the cocoon may be added to the cosmetic in an amount of 0.1% to 10% by weight based on the total liquid weight of the cosmetic composition, and the total amount of the cosmetic composition It may be added to the cosmetic in an amount of 0.001 to 5% by weight, preferably 0.01 to 3% by weight, based on the dry weight.

In addition, the present invention can provide a food composition for preventing or improving skin inflammation and a health functional food comprising a papain enzyme decomposition product for an ultrasonic extract of a cocoon as an active ingredient.

In the present invention, the term "food composition" refers to a food that provides a better health condition by acting advantageously on one or more functions of the organism, regardless of the nutrients provided to the subject ingesting the same. Consequently, the food composition can be used for the prevention, improvement or treatment of diseases or disease-causing factors.

Accordingly, the term "food composition" of the present invention may be used as a synonym for functional food or food or pharmaceutical food for a specific nutritional purpose.

The food composition of the present invention may additionally contain a conventional pharmaceutically acceptable carrier or excipient in addition to the papain enzyme decomposition product for the ultrasonic extract of the cocoon of the present invention, and in addition, such as a binder, a decomposition agent, a coating agent, a lubricant, etc. It can be formulated and formulated with various additives commonly used pharmaceutically.

In one embodiment of the present invention, the papain enzyme decomposition product for the ultrasonic extract of the cocoon may be included in an amount of 0.001 to 99% by weight based on the total weight of the composition.

The food composition of the present invention may contain, as an additional component, various flavoring agents or natural carbohydrates, as in a conventional food composition, in addition to containing a papain enzyme decomposition product to the ultrasonic extract of the cocoon of the present invention.

Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents can be advantageously used as natural flavoring agents (taumatin), stevia extracts (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

In addition, the food composition of the present invention can be preferably formulated as a food composition, including at least one food pharmaceutically acceptable or pharmaceutically acceptable carrier in addition to the papain enzyme decomposition product for the ultrasonic extract of the cocoon of the present invention. .

The formulation form of the food composition may be a tablet, capsule, powder, granule, liquid, pill, liquid, syrup, juice, suspension, emulsion, or drop. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The food composition of the present invention formulated in the above manner may be used as a functional food or added to various foods.

Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. There is this.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), in addition to the papain enzyme decomposition product for the ultrasonic extract of the cocoon, Pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages.

The papain enzyme decomposition product for the ultrasonic extract of the cocoon, the active ingredient of the present invention, has almost no side effects such as chemicals, and can be safely used even when taken for a long time for the purpose of improving or preventing skin inflammation.

Accordingly, the present invention provides a health functional food for preventing or improving skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for the ultrasonic extract of the cocoon of the present invention.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving skin inflammation.

In the present invention, the term'health functional food' refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act, and controls nutrients for the structure and function of the human body. It means ingestion for the purpose of obtaining a useful effect for health use such as physiological action.

The health functional food of the present invention may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods for food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards.

Examples of the items listed in the "Food Additives Code" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned.

For example, in the health functional food in the form of a tablet, a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives is granulated by a conventional method, and then a lubricant, etc. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like, if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling the active ingredients of the present invention into ordinary hard capsules, and soft capsules are prepared by mixing the active ingredients of the present invention with additives such as excipients, such as gelatin. It can be prepared by filling in a capsule base. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

Ring-shaped health functional foods can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc. by conventionally known methods, and can be coated with white sugar or other coating agents if necessary, Alternatively, the surface may be coated with a material such as starch or talc.

The health functional food in the form of granules can be prepared in granular form by a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., by a known method, and may contain flavoring agents, flavoring agents, etc. have.

The health functional food may be beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements.

In addition, the present invention provides a pharmaceutical composition for preventing or treating skin inflammation, comprising as an active ingredient a papain enzyme decomposition product for an ultrasonic extract of a cocoon.

The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier, and the "pharmaceutically acceptable" means that it does not significantly stimulate an organism and does not inhibit the biological activity and properties of the administration active substance. do.

The term "prevention" as used in the present invention refers to any action that suppresses the symptoms of skin inflammation or delays the progression by administration of the composition of the present invention.

The term "treatment" as used in the present invention refers to any action that improves or beneficially alters the symptoms of skin inflammation by administration of the composition of the present invention.

The term "improvement" as used herein refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example a symptom.

As used herein, the term "administering" means providing a given composition of the present invention to a subject by any suitable method. At this time, the individual refers to all animals, such as humans, monkeys, dogs, goats, pigs, mice, etc., having diseases in which the symptoms of skin inflammation can be improved by administering the composition of the present invention.

In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which is the type of disease, severity, activity of the drug, and Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used at the same time, and other factors well known in the medical field.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the papain enzyme digestion product for the ultrasonic extract of the cocoon of the present invention, and the adjuvant may be an excipient, a disintegrant, Sweetening agents, binders, coating agents, expanding agents, lubricants, lubricants or flavoring agents may be used.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition, including at least one pharmaceutically acceptable carrier in addition to the papain enzyme digestion product for the ultrasonic extract of the cocoon of the present invention.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Further, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the papain enzyme decomposition product for the ultrasonic extract of the cocoon of the present invention may be included in an amount of 0.001 to 99% by weight based on the total weight of the composition.

In addition, the skin inflammation in the present invention may include, without limitation, all skin inflammatory diseases accompanying an inflammatory reaction occurring in the skin, and atopic dermatitis, contact dermatitis, seborrhea ) And may be any one selected from the group consisting of acne.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

< Example 1>

Cocoon Papain (papain) enzyme resolvent Produce

<1-1> By concentration Sodium hydroxide Preparation of protein extract derived from cocoon using solution

100 mL of 0.1, 0.25, 0.50, 1N NaOH solution was added to 1 g of cocoon and reacted for one day, and then the protein content of the extract derived from the cocoon was determined by time in a DC protein kit (Bio-Rd, Hercules, CA, USA) It was measured using, and the extraction yield was measured using the following calculation formula.

Extraction yield (%) = protein content (g) / sample weight (g) X 100

In general, a high concentration of organic solvent is used to produce silk proteins derived from cocoons. However, a high concentration of organic solvent has a problem that can reduce the activity of protein and cause denaturation, so studies to reduce the amount of the organic solvent have been attempted. Accordingly, the present inventors obtained a protein extract from a cocoon using sodium hydroxide solutions having different concentrations at low concentrations as described above and analyzed the yield thereof.

As a result, as shown in Fig. 1, it was found that the higher the concentration of sodium hydroxide, the higher the concentration of the extracted protein. In addition, while the extraction was performed for up to 6 hours, it was found that the content of the extracted protein rapidly increased, but when the time exceeded 6 hours, the protein extracted content decreased as time passed. These results can also be known through the results of Table 1 above.

That is, as shown in Table 1, it can be seen that the extraction yield is 50% or less at a low salt concentration, and at a higher concentration, the yield increases up to 80%. It was also found that the extraction time was also shortened.

<1-2> Preparation of protein extract derived from cocoon using ultrasonic treatment

Furthermore, the present inventors applied ultrasonic treatment as a method for obtaining a protein extract with high yield from a cocoon while using a low concentration of organic solvent (sodium hydroxide solution). To this end, 500 mL of 0.1N NaOH and 0.25N NaOH solution was added to 5 g of the cocoon from which impurities were removed, and ultrasonic treatment was performed, and the ultrasonic treatment conditions were 20 kHz, 750W, 80% amplitude, and 20s/20s pulse. , Treated at 25°C. In addition, the content of the extracted protein was measured using a DC protein kit.

As a result, as shown in FIG. 2, when ultrasonic treatment was performed using a 0.25N sodium hydroxide solution, it was found that the protein extraction content was significantly increased compared to the control group not subjected to ultrasonic treatment. In addition, when ultrasonication was performed using 0.1 N sodium hydroxide solution, the difference in protein content before and after the ultrasonic treatment was found to be greater. In addition, as a result of analysis of the change in extraction content by reaction time, in the case of 4 hours of sonication, when comparing the protein extraction content, the group that was sonicated in 0.1N sodium hydroxide solution was 6 like the group that was not sonicated in 0.25N sodium hydroxide solution. As a similar protein content of mg/mL or more was shown, the following enzymatic reaction was performed after fixing by treating ultrasonic waves for 4 hours in a 0.1 N sodium hydroxide solution, which is a condition of a sodium hydroxide solution having a lower concentration.

<1-3> Papain (papain) enzyme treatment

The cocoon protein extract obtained by ultrasonic treatment for 4 hours in a 0.1 N sodium hydroxide solution, which is the optimal ultrasonic treatment condition confirmed through the experiment of <1-2>, was titrated to pH 7.0 with 6N HCl, and then 0.5% papain ( papain) enzyme was added and the enzyme was reacted for 2 days. After the reaction was completed, it was heat-treated at 100° C. for 10 minutes to prepare a papain enzyme hydrolyzate of cocoon.

< Example 2>

Molecular weight (MW) measurements of papain enzyme degradation product of a cocoon produced from the cocoon of the invention papain (papain) protein molecular weight measurement of the enzyme degradation product of Example 1 is arranged the detector diode by using a gel permeation chromatography (GPC) technique Was measured with the Ultimate 3000 system (Thermo Fisher, Somerset, NJ, USA) equipped. The column was a Protein KW-802.5 (ID 8 mm × 300 mm, 4 μm) column, the sample was filtered through a 0.45 μm PVDF syringe filter, the flow rate was 1 min/mL, the mobile phase was 50 mM sodium phosphate buffer containing 0.3 M NaCl, wavelength Was measured at 220 nm. As a molecular weight standard, β-Amylase (200kDa), alcohol dehydrogenase (150kDa), albumin (66kDa), carbon anhydrase (29kDa), cytochrome c (12.4kDa), aprotin (6.5kDa), simatostatin (1.63) kDa) was used.

As a result, as shown in Fig. 3, the molecular weight change analysis result of the reaction product enzymatically reacted by adding papain enzyme to the extract obtained for each sonication time in 0.1 N sodium hydroxide solution is as shown in Figure 3, the protein contained in the extract according to the papain enzyme treatment. This decomposition was found to reduce the size of the molecular weight. Specifically, the molecular weight of the protein extract derived from cocoon that was treated only with ultrasonication was mainly detected to be proteins with a molecular weight of 5 kDa or more, whereas the addition of papain enzyme was found to be less than 5 kDa. It was found that the content of peptides having a molecular weight of 1.4 kDa increased by decomposition into protein.

< Example 3>

Cocoon of the present invention Papain (papain) enzyme Decomposition Analysis of skin inflammation relief effect

Whether the effect of reducing skin inflammation with the enzyme digestion product of the cocoon papain of the present invention prepared in Example 1 was analyzed through the following experiment.

① COX- 2(Cyclooxygenase-2) expression Evaluation of inhibitory efficacy

Starvation was performed for 12 hours by incubating the mouse epithelial cell line (JB6 P+) in an animal cell culture dish (100 pi dish) for 24 hours and then replacing it with a medium (DMEM) not containing fetal bovine serum (FBS). After starvation, each sample was treated in a medium according to the concentration for 1 hour, and 30 kJ/m2 of sunlight-like ultraviolet rays (solar UV, sUV) were treated. After treatment with solar UV, sUV, the protein was extracted into the cell incubator after 3 hours and 30 minutes, and the expression level of COX-2 in the protein was measured using Western blot.

② Prostaglandin-E2 ( ProstaGlandin -E2, PGE2 ) Productivity analysis

Starvation was performed for 12 hours by culturing the mouse epithelial cell line (JB6 P+) in an animal cell culture dish (100 pi dish) for 24 hours and then replacing it with a medium (DMEM) containing no fetal bovine serum (FBS). After starvation, each sample was treated in the medium according to the concentration for 1 hour, and 30 kJ/m2 of sunlight-like ultraviolet rays (solar UV, sUV) were treated, and then in a cell incubator (incubator). And incubated for 5 hours. Thereafter, the medium was harvested and the amount of PGE2 was measured with a PGE2 ELISA kit (Cat. # 514010, CAYMAN).

③ Inflammatory signaling system (Inflammatory signaling pathway) Evaluation of phosphorylation inhibitory efficacy

Starvation was performed for 12 hours by incubating the mouse epithelial cell line (JB6 P+) in an animal cell culture dish (100 pi dish) for 24 hours and then replacing it with a medium (DMEM) containing no fetal bovine serum (FBS). After starvation, each sample was treated in the medium according to the concentration for 1 hour, and 30 kJ/m2 of sunlight-like ultraviolet rays (solar UV, sUV) were treated, and then in a cell incubator (incubator). After 30 minutes of insertion, proteins were extracted from the cells, and then Western blot was performed to measure the phosphorylation of factors related to the inflammatory signaling pathway in the protein.

As a result of the analysis, as shown in FIG. 4, the extract derived from cocoon obtained by treatment with only ultrasonic waves showed insufficient inhibition of COX-2 expression, whereas the expression of COX-2 in the hydrolyzate obtained by adding papain enzyme to the ultrasonic extract. This was shown to be suppressed, and when the addition amount of papain was tested by concentration at 1:50, 1:100, and 1:200, it was found that COX-2 expression was suppressed in a concentration-dependent manner. On the other hand, it was found that the inhibition of the expression of COX-2 was insufficient in the group treated with other types of enzymes such as pancreatin, protease and bromelain. In addition, it was found that the production of prostaglandin-E2 was also inhibited in the hydrolyzate obtained by adding papain enzyme.

Therefore, through these results, the present inventors found that in order to effectively suppress the expression of COX-2, which is the main cause of skin inflammation, the hydrolyzate obtained by treating the papain enzyme in the ultrasonic cocoon extract obtained by ultrasonic treatment is the most effective. Could know.

In addition, the results of Fig. 5 showed that the papain enzyme hydrolyzate of the ultrasonic cocoon extract of the present invention effectively inhibits phosphorylation of ERK, a major factor in the inflammatory signaling system, while silkworms obtained by treating only ultrasonic waves The ultrasonic cocoon extract showed insufficient inhibitory effect on phosphorylation of ERK, and the group treated with pancreatin, protease and bromelain enzymes also showed insufficient effect.

Based on the above results, the papain enzyme hydrolyzate of the ultrasonic cocoon extract of the present invention can effectively inhibit both the activation of the inflammatory signaling system and the expression of COX-2, which are the main causes of skin inflammation, thereby improving skin inflammation. , It was found that it can be used as a material for treatment or prevention.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (13)

Adding 0.1 N sodium hydroxide solution to the cocoon, and ultrasonicating the cocoon at a temperature of 25° C. at 20 kHz for 4 hours to obtain an ultrasonic extract; And
Including the step of hydrolyzing by adding papain enzyme to the ultrasonic extract,
Method for producing a papain enzyme digestion product of an ultrasonic cocoon extract having an activity to inhibit or improve skin inflammation. delete delete Contains papain enzyme digestion product for ultrasonic extract of cocoon as an active ingredient,
As for the papain enzyme decomposition product for the ultrasonic extract of the cocoon, 0.1 N sodium hydroxide solution was added to the cocoon, and ultrasonically treated at a temperature of 25° C. at 20 kHz for 4 hours to obtain an ultrasonic extract. Characterized in that obtained by adding and hydrolyzing papain enzyme to the extract,
Cosmetic composition for improving skin inflammation. delete The method of claim 4,
The papain enzyme digestion product for the ultrasonic extract of the cocoon is characterized in that it inhibits COX-2 (Cyclooxygenase-2) inhibition, PGE2 (Prostaglandin E2) inhibition, or ERK (extracellular-signal-regulated kinase) phosphorylation. , Cosmetic composition for improving skin inflammation. Contains papain enzyme digestion product for ultrasonic extract of cocoon as an active ingredient,
As for the papain enzyme decomposition product for the ultrasonic extract of the cocoon, a 0.1 N sodium hydroxide solution was added to the cocoon and ultrasonicated at a temperature of 25° C. at 20 kHz for 4 hours to obtain an ultrasonic extract, and the ultrasonic wave Characterized in that obtained by adding and hydrolyzing papain enzyme to the extract,
Health functional food for preventing or improving skin inflammation. delete The method of claim 7,
The papain enzyme digestion product for the ultrasonic extract of the cocoon is characterized in that it inhibits COX-2 (Cyclooxygenase-2) inhibition, PGE2 (Prostaglandin E2) inhibition, or ERK (extracellular-signal-regulated kinase) phosphorylation. , Health functional food for preventing or improving skin inflammation. Contains papain enzyme digestion product for ultrasonic extract of cocoon as an active ingredient,
As for the papain enzyme decomposition product for the ultrasonic extract of the cocoon, a 0.1 N sodium hydroxide solution was added to the cocoon and ultrasonicated at a temperature of 25° C. at 20 kHz for 4 hours to obtain an ultrasonic extract, and the ultrasonic wave Characterized in that obtained by adding and hydrolyzing papain enzyme to the extract,
A pharmaceutical composition for preventing or treating skin inflammation. delete The method of claim 10,
The papain enzyme digestion product for the ultrasonic extract of the cocoon is characterized in that it inhibits COX-2 (Cyclooxygenase-2) inhibition, PGE2 (Prostaglandin E2) inhibition, or ERK (extracellular-signal-regulated kinase) phosphorylation. , A pharmaceutical composition for preventing or treating skin inflammation. The method of claim 10,
The skin inflammation is atopic dermatitis (atopic dermatitis), contact dermatitis (contact dermatitis), seborrheic dermatitis (seborrhea), characterized in that selected from the group consisting of acne, a pharmaceutical composition for preventing or treating skin inflammation.

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