KR102169046B1 - Antiinflammatory composition comprising enzyme treated larva of Protaetia brevitarsis seulensis - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing an enzyme treatment product of white spotted flower radish ( Protaetia brevitarsis seulensis) larvae. The enzyme-treated product of the white spotted radish increases various useful components by simply hydrolyzing the larvae of the white spotted radish with a flavozyme enzyme, thereby increasing NO (nitric oxide) and iNOS (inducible nitric oxide). synthase), TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), and other inflammation-related factors. Therefore, the enzyme treatment product of the white spotted radish is inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis. , Cystitis, nephritis, neuritis, etc. can be easily used for prevention, improvement or treatment.

Description

Antiinflammatory composition comprising enzyme treated larva of Protaetia brevitarsis seulensis}

The present invention relates to an anti-inflammatory composition containing an enzyme treatment product of white spotted flower radish ( Protaetia brevitarsis seulensis) larvae. More specifically, the present invention inhibits the production of inflammation-related factors such as NO (Nitric oxide), iNOS (inducible nitric oxide synthase), TNF-α (tumor necrosis factor-α), and IL-1β (interleukin-1β). Inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia , Pancreatitis, sepsis, cystitis, nephritis, neuritis, etc., to a composition for anti-inflammatory excellent in prevention, improvement or treatment efficacy.

Inflammation is one of the defense responses of biological tissues to certain stimuli, and refers to a complex lesion that causes three types of tissue deterioration, circulatory disorders and effusion, and tissue proliferation. The causes are mechanical injury, physical factors such as temperature, radiation, chemical factors such as poisons, and parasites such as bacterial infection, among which bacteria are the most common. In addition to these main causes, its occurrence is complicated by various incidental factors and predisposition or immunity of the individual.

In addition, the inflammatory reaction can be said to be one of the most important reactions among pathological mechanisms according to the host's defense mechanism against wounds and microbial infections. Macrophages are the most representative immune cells that regulate this inflammatory response, and activated macrophages are TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), PGE2 (prostaglandin E2), and NO (nitric oxide). ), ROS (reactive oxygen species), and the like (Laskin, DL et al., 2011).

Accordingly, the inventors of the present invention confirmed that the enzyme-treated product obtained by enzymatic treatment of the white spotted radish has excellent anti-inflammatory efficacy compared to the non-enzyme-treated group while studying various physiological activities of the white-spotted radish. It was found that the present invention could be easily used as a therapeutic agent.

Korean Patent Registration No. 10-1382400 (Name of the invention: composition for preventing and treating inflammatory diseases containing white spotted radish as an active ingredient, Applicant: Republic of Korea, registration date: April 1, 2014) Republic of Korea Patent Publication No. 10-2018-0100814 (Name of the invention: protein hydrolyzate of larvae of Protaetia brevitarsis, preparation method thereof and composition containing the same, Applicant: Republic of Korea, Publication date: September 12, 2018 Work)

It is an object of the present invention to provide an anti-inflammatory composition containing an enzyme treatment product of white spotted flower radish ( Protaetia brevitarsis seulensis) larvae. More specifically, the object of the present invention is to prevent the generation of inflammation-related factors such as NO (Nitric oxide), iNOS (inducible nitric oxide synthase), TNF-α (tumor necrosis factor-α), and IL-1β (interleukin-1β). Inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis , Pneumonia, pancreatitis, sepsis, cystitis, nephritis, neuritis, etc. to provide an excellent anti-inflammatory composition for preventing, improving or treating.

The present invention relates to an anti-inflammatory composition containing an enzyme treatment product of white spotted flower radish ( Protaetia brevitarsis seulensis) larvae.

The enzyme-treated product of the white spotted radish larvae is a low molecular weight of 3 kDa or less obtained by filtering the liquid obtained by treating the powder obtained by drying the white spotted radish larva with flavozyme using an ultrafiltration membrane (centrifugal filter). It is characterized by being a protein concentrate.

The enzyme-treated product of the white spotted radish larva is one from the group consisting of NO (nitric oxide), iNOS (inducible nitric oxide synthase), TNF-α (tumor necrosis factor-α), and IL-1β (interleukin-1β). It is characterized in that it suppresses the production of the above-selected inflammation-related factors.

The present invention can also provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the enzyme-treated product of white spotted radish larva.

The inflammatory diseases include inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, cystitis, nephritis and neuritis. It may be selected from the group consisting of.

In another aspect, the present invention also relates to a health functional food for the prevention or improvement of inflammatory diseases containing the enzyme treatment product of white spotted flower radish larva.

In addition, the present invention provides an anti-inflammatory cosmetic composition comprising the enzyme-treated product of the larvae of the white spotted flower radish as an active ingredient.

Hereinafter, the present invention will be described in detail.

The enzyme-treated product of the white spotted radish larvae is a low molecular weight of 3 kDa or less obtained by filtering the liquid obtained by treating the powder obtained by drying the white spotted radish larva with flavozyme using an ultrafiltration membrane (centrifugal filter). It is characterized by being a protein concentrate.

At this time, the enzyme treatment product of the white spotted radish larva may be prepared including the following steps.

Even more preferably,

(Step 1) adding white spotted radish larva powder to water, physiological saline, phosphate buffered physiological saline or sodium phosphate buffer to obtain a powdery liquid;

(Second step) reacting the powdered liquid phase at 80 to 100° C. to inactivate autologous enzymes;

(3rd step) adding a flavozyme to the powdered liquid in which the autoenzyme is inactivated, followed by enzymatic reaction;

(Step 4) After completion of the enzymatic reaction, centrifuging to collect the supernatant and reacting at 80 to 100° C. for 10 to 30 minutes to inactivate the enzyme activity of flavozyme;

(Fifth step) filtering and sterilizing the reaction solution in which the enzyme activity is inactivated; And,

(Sixth step) obtaining a low molecular weight protein concentrate having a molecular weight of 3 kDa or less using an ultrafiltration membrane (centrifugal filter);

Through it can be obtained the enzyme treatment of the white spotted flower radish larvae of the present invention.

Even more preferably,

(Step 1) adding white spotted radish larva powder to water, physiological saline, phosphate buffered physiological saline or sodium phosphate buffered solution in an amount of 5 to 15% (w/v) to obtain a powdery liquid;

(Second step) inactivating the autologous enzyme by reacting the powdered liquid at 80 to 100° C. for 10 to 30 minutes;

(3rd step) adding a flavozyme to 2-6% (w/v) to the powder liquid in which the autoenzyme is inactivated, and enzymatic reaction at 35-28° C. for 16-20 hours;

(Step 4) After completion of the enzymatic reaction, centrifugation at 800 to 1500 rpm for 10 to 20 minutes to collect the supernatant and reacting at 80 to 100° C. for 10 to 30 minutes to inactivate the enzyme activity of flavozyme;

(Step 5) After obtaining a supernatant by centrifuging the reaction solution inactivated enzyme activity at 10000-15000 rpm for 10-20 minutes, filtering and sterilizing using a syringe filter; And,

(Sixth step) obtaining a low molecular weight protein concentrate having a molecular weight of 3 kDa or less by centrifuging at 4000 to 6000 rpm for 0.5 to 2 hours using an ultrafiltration membrane (centrifugal filter);

Through it can be obtained the enzyme treatment of the white spotted flower radish larvae of the present invention.

The powder of the white spotted radish larvae is a powdered white spotted radish that has been fasted for 2 to 3 days, and dried by freeze drying, hot air drying or natural drying (dried at room temperature at 20 ~ 30℃). It can be obtained by powdering the larva. At this time, the conditions for drying the larva can be appropriately controlled, and preferably can be carried out for 1 to 5 days, and the white spotted radish larvae are 5~ at 110~120℃ and 1.0~1.2 kgf/㎠ after fasting. It is preferable to use after sterilization by reacting for 20 minutes.

In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the enzyme-treated product of the white spotted flower radish larva as an active ingredient.

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

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is from 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

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

The present invention can provide a health functional food for preventing or improving inflammatory diseases comprising the enzyme-treated product of the white spotted flower radish larva as an active ingredient.

The health functional food may contain food supplementary additives that are food wise acceptable. The health functional food of the present invention includes forms such as tablets, capsules, pills or liquids, and foods to which the extract of the present invention can be added include, for example, various drinks, meats, sausages, bread, candy, There are snacks, noodles, ice cream, dairy products, soups, ionized drinks, beverages, alcoholic beverages, gum, tea and vitamin complexes.

In another aspect, the present invention can provide an anti-inflammatory cosmetic composition comprising the enzyme treatment product of the white spotted flower radish larva as an active ingredient. The formulation of the cosmetic composition is not greatly limited, but preferably skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation , Essence, nutrition essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser. Ingredients included in the cosmetic composition of the present invention may include all of the ingredients generally used in cosmetics in addition to the enzyme treatment product of the white spotted radish larva of the present invention as an active ingredient. For example, it may contain general auxiliary ingredients such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins and fragrances.

The present invention relates to an anti-inflammatory composition containing an enzyme treatment product of white spotted flower radish ( Protaetia brevitarsis seulensis) larvae. The enzyme-treated product of the white spotted radish increases various useful components by simply hydrolyzing the larvae of the white spotted radish with a flavozyme enzyme, thereby increasing NO (Nitric oxide) and iNOS (inducible nitric oxide). synthase), TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), and other inflammation-related factors. Therefore, the enzyme treatment product of the white spotted radish is inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis. , Cystitis, nephritis, neuritis, etc. can be easily used for prevention, improvement or treatment.

As a prior art related to the present invention, Korean Patent No. 10-1382400 discloses that the white spotted radish larva has an effect of treating inflammation, but the enzyme treatment of white spotted radish larva of the present invention is more anti-inflammatory than the larva before enzyme treatment. It is confirmed that the effect is excellent. In addition, although Korean Patent Application Publication No. 10-2018-0100814 discloses a process of manufacturing an enzyme-treated product of white spotted radish larva, the anti-inflammatory effect of such an enzyme-treated product is not disclosed at all. Therefore, compared to these prior arts, the technical differences and superiority of the present invention are confirmed.

1 shows the results of confirming the state before and after enzymatic treatment of the white spotted radish larva powder liquid through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and staining with Coomassie brilliant blue.
Figure 2 shows the result of confirming the hydrolysis rate of the enzyme-treated product of the white spotted radish larva of the present invention.
3 shows the results of confirming the cytotoxicity of the enzyme-treated product of the white spotted radish larva of the present invention through the MTS assay.
Figure 4 shows the results of confirming the effect of reducing the secretion amount of NO (Nitric oxide) of the enzyme treatment product of the white spotted radish larva of the present invention in LPS (Lipopolysaccaride)-treated mouse macrophage Raw 264.7.
5 shows the results of confirming the effect of inhibiting the production of iNOS of the enzyme-treated product of the white spotted radish larva of the present invention in Raw 264.7, which is a mouse macrophage treated with LPS (Lipopolysaccaride).
6 shows the results of confirming the effect of inhibiting the production of TNF-α of the enzyme treatment product of the white spotted radish larva of the present invention in Raw 264.7, which is a mouse macrophage treated with LPS (Lipopolysaccaride).
7 shows the results of confirming the effect of inhibiting the production of IL-1β of the enzyme-treated product of the white spotted radish larva of the present invention in LPS (Lipopolysaccaride)-treated mouse macrophages Raw 264.7.

Hereinafter, the present invention will be described in more detail through examples. The following examples are presented only to illustrate the present invention, and the present invention is not limited by the following examples.

<Example 1. Preparation of enzyme-treated product of white spotted radish larva>

Example 1-1. Pretreatment of white-spotted radish larva

After fasting the larvae of white spotted flowers for 3 days, the feces were separated using a tray and washed with running water. After spreading the nonwoven fabric in the sterilizer container, the white spotted radish larva was put and sterilized for 10 minutes in a high temperature/high pressure sterilizer (115° C., 1 kgf/㎠). The sterilized white spotted radish larva was stored in an ultra-low temperature freezer for 24 hours, lyophilized for 72 hours, and then pulverized with a multifunctional grinder to obtain powder of white spotted radish larva.

Example 1-2. Enzymatic treatment of white spotted radish larva and obtaining low-molecular protein

The white spotted radish larva powder obtained in Example 1-1 was suspended in 10 mM sodium phosphate buffer (pH 7.0) to prepare a 10% (w/v) substrate liquid.

The substrate liquid was heat-treated at 90°C for 20 minutes by shaking culture method to inactivate the autologous enzyme, and after adding the protein hydrolase flavourzyme to 4% (w/v) compared to the substrate liquid, shaking culture at 37°C for 18 hours Then, the enzyme was treated.

The enzyme-treated liquid phase was centrifuged at 1,000 rpm for 15 minutes, and then the supernatant was collected, and the supernatant was heat-treated at 90° C. for 20 minutes to inactivate the enzyme. The enzyme-inactivated liquid phase was centrifuged again at 13000 rpm for 15 minutes to obtain a supernatant, and then filtered and sterilized with a 0.45 μm syringe filter.

Finally, the filtrate obtained by the syringe filter using an ultrafiltration membrane (centrifugal filter) was centrifuged at 5,000 rpm for 1 hour to finally obtain a low molecular weight protein concentrate having a molecular weight of 3 kDa or less. It was used as an enzyme treatment product.

<Example 2. Confirmation of protein hydrolysis properties of enzyme-treated products of white spotted radish larva>

Example 2-1. Enzyme treatment confirmation using protein electrophoresis (SDS-PAGE)

In order to confirm the protein size change and protein degradation of the white spotted radish larva due to the enzyme treatment, the supernatant obtained before ultrafiltration membrane filtration after treatment with the flavourzyme enzyme in the process of obtaining the enzyme treatment of Example 1-2 was used as a test group, and enzyme The powder liquid phase before treatment was set as a control.

After quantifying the protein concentration in the liquids of the control and test groups by BCA protein assay, electrophoresis was performed by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and the protein status was confirmed after staining with Coomassie brilliant blue.

The results are shown in Fig. 1, in which proteins of various sizes were confirmed in the liquid phase (NE) of the white spotted radish larvae as a control, but large-sized proteins were hardly identified in the test group treated with the flavourzyme enzyme. It can be confirmed that bands appear only in small sizes due to hydrolysis.

Example 2-2. Identification of small molecule proteins using the TNBS (trinitrobenzensulfonic acid) method

After adding 1M sodium borate buffer to 20㎍/mL of the enzyme treatment product finally obtained in Example 1-2, it was mixed with 5mM trinitrobenzensulfonic acid and reacted at 37°C for 2 hours. Thereafter, 18 mM sodium sulfite and 2M monobasic sodium phosphate were added to terminate the reaction, and the absorbance was measured at 335 nm. At this time, the control group was a white spotted radish larva powder liquid before enzyme treatment.

The results are shown in Fig. 2, and it can be seen that the hydrolysis rate of each enzyme-treated product increased by about 1.8 times compared to the liquid phase of the white spotted radish larva powder before the enzyme treatment.

<Example 3. Cytotoxicity of the enzyme-treated product of white spotted radish larva>

To confirm cytotoxicity, MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- using mouse macrophage Raw264.7 cells. tetrazolium, inner salt) assay was performed. Raw264.7 cells were prepared in DMEM medium containing antibiotics (100 units/mL penicillin, 100 ㎍/mL streptomycin) and 10% (v/v) fetal bovine serum (FBS) in a 37°C, 5% CO ₂ incubator. It was cultured and used in the experiment.

To perform the MTS assay, 8 × 10 ⁴ cell/well cells were placed in 100 µl medium in a 96-well culture vessel and then dispensed, and then the enzyme-treated product of white spotted radish larva was treated in a concentration-dependent manner for 24 hours. After that, 10 µl of MTS reagent (CellTiter 96 ^® AQueous One Solution Reagent, Promega, USA) was added and reacted for 3 to 4 hours to check the color change, and absorbance measurement at 450 nm using a multi detecter (Beckman DTX8800). Confirmed.

The results are shown in Fig. 3, and it was confirmed that the enzyme-treated product of the white spotted radish larva had little toxicity to Raw264.7 cells up to the maximum concentration of 500 µg/ml.

<Example 4. Anti-inflammatory activity assay for enzyme-treated products of white spotted radish larva>

Example 4-1. NO production inhibitory effect

Raw 264.7 cells are known to produce NO (Nitric oxide) when inducing inflammation and secrete it extracellularly. Therefore, in order to confirm the anti-inflammatory effect of the enzyme treatment of the white spotted radish larva, it was treated on Raw264.7 cells and the amount of NO secreted was confirmed.

To this end, 100 µl of 8 × 10 ⁴ cell/well cells were dispensed into 96 wells, and then the enzyme-treated product of white spotted radish larva was treated in a concentration-dependent manner for 24 hours. After that, to induce inflammation, lipopolysaccharide (LPS) was treated with 100 ng/ml and cultured for 24 hours.

Next, according to the method of the NO detection kit (intron), 100 µl of the supernatant of the cultured cells was used in the experiment to determine how much the amount of NO secreted by LPS was reduced by the enzyme treatment of the white spotted radish larva. It was confirmed by measuring absorbance at 550nm wavelength of multi detecter (Beckman DTX8800).

The results are shown in Fig. 4, and as a result, it can be seen that the concentration-dependent amount of NO secreted in the enzyme-treated product of the white-spotted radish larvae decreases at 50 µg/ml.

Example 4-2. Inhibitory effect of iNOS on protein expression

iNOS does not normally exist in cells, but once induced, it produces a large amount of NO for a long time, and it is known that the generated NO promotes the inflammatory response and promotes the biosynthesis of inflammatory mediators to intensify inflammation. Therefore, it can be confirmed that the anti-inflammatory effect occurred by confirming the decrease in the protein level of iNOS in Raw 264.7 cells. In addition, such inhibition of NO and iNOS leads to a decrease in the protein level of COX-2, which is one of the factors that increase proinflammatory cytokines such as TNF-α and IL-6, and thus anti-inflammatory effects can be expected.

Thus, the protein expression of iNOS was measured by western blot using macrophages treated with enzyme-treated product of white spotted radish larva.

After harvesting raw 264.7 cells, centrifugation was performed, the supernatant was discarded, and the cell pellet was collected. The cells were lysed using RIPA buffer and then centrifuged (14,000 rpm, 20 min) to collect the supernatant. After quantifying the protein with the BCA protein assay kit (Pierce, Rockford, IL, USA), the same amount of the enzyme-treated product of white spotted radish larva was separated by sodium dodecyl sulfate-polyacrlyamide gel electrophoresis (SDS-PAGE), and then PVDF It was transferred to the membrane (Bio-Rad Laboratories, Inc., USA).

PVDF was reacted with 5% skim milk for 1 hour to block reactivity to non-specific proteins, reacted with anti-iNOS antibody, and then reacted with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour. Between each reaction, it was washed 3 times with 0.05% TBST for 10 minutes each. Subsequently, the corresponding protein band for the antibody was confirmed using the ECL kit (Amersham Pharmacia Biotech, UK).

As a result, as shown in Figure 5, it can be seen that the amount of iNOS protein expression increased by LPS in Raw 264.7 cells is decreased in a concentration-dependent manner in the enzyme (flavourzyme) treatment, through which the enzyme treatment product of the white spotted radish larva produces iNOS. It can be seen that it inhibits.

Example 4-3. TNF-α, IL-1β inhibiting protein production

Macrophages are immune cells that appear in all tissues in the animal body, and they remove bacteria or foreign substances by phagocytosis, and play an important role in the initial inflammatory reaction by secreting inflammatory mediators such as IL-1β, IL-6, and TNF-α. In particular, TNF-α plays an important role in the inflammatory response and is secreted from macrophages and mast cells, and plays an important role in intrinsic immunity as a major mediator of the LPS response and is also related to chronic inflammatory reactions. IL-1β has been reported to activate T-cell activation, B-cell maturation, and NK cell activity.

Accordingly, the amount of TNF-α and IL-1β produced in the cell culture solution was measured using an ELISA kit. Raw 264.7 cells were adjusted to 8x10 ⁵ cells/ml using DMEM medium, inoculated into 6 well plates, and cultured in a 5% CO2 incubator for 24 hours. White spotted radish larvae were treated with low molecular weight proteins (50, 100, 250, 500 ㎍/ml) in a concentration-dependent manner, and then treated with 100 ng/ml of lipopolysaccharide (LPS) to induce inflammation and cultured for 24 hours. Thereafter, a culture medium was taken and TNF-α and IL-1β were measured.

As a result, the production of TNF-α and IL-1β increased through the treatment of LPS in Raw 264.7 cells as shown in FIGS. 6 and 7 were all about 500 μg/ml as a result of treatment with the enzyme treatment of white spotted radish larva. It is confirmed to exhibit a 50% production inhibitory effect.

<Formulation Example 1. Pharmaceutical formulation>

200 g of the flavourzyme treated product (final reactant of Example 1-2) of the white spotted radish larva of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. After adding a 10% gelatin solution to this mixture, it was pulverized and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.

<Formulation Example 2. Food Manufacture>

Formulation Example 2-1. Preparation of cooking seasoning

The flavourzyme-treated product of the white spotted radish larva of the present invention was added to the cooking seasoning in an amount of 1% by weight to prepare a cooking seasoning for health promotion.

Formulation Example 2-2. Manufacture of flour food

The flavourzyme-treated product of the white spotted radish larva of the present invention was added to flour in an amount of 0.1% by weight, and bread, cakes, cookies, crackers, and noodles were prepared using this mixture to prepare food for health promotion.

Formulation Example 2-3. Preparation of soups and gravies

The flavourzyme-treated product of the white spotted radish larva of the present invention was added in an amount of 0.1% by weight to soup and broth to prepare soups and juices for health promotion.

Formulation Example 2-4. Manufacture of dairy products

The flavourzyme treated product of the white spotted radish larva of the present invention was added to milk in an amount of 0.1% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

Vegetable juice for health promotion was prepared by adding 0.5 g of the flavourzyme treatment product of the white spotted radish larva of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Fruit juice manufacturing

Fruit juice for health promotion was prepared by adding 0.1 g of the flavourzyme treatment product of the white spotted radish larva of the present invention to 1,000 ml of apple juice or grape juice.

<Cosmetic formulation example 1. Preparation of flexible lotion>

As shown in the composition of Table 1 below, a flexible lotion (skin, 100 g) containing the flavourzyme treatment of the white spotted radish larva of the present invention was prepared according to a conventional method.

Raw material Content (g) Flavourzyme treatment of white-spotted radish larva 0.3 glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Polyoxyethylene (60) hardened castor oil 1.0 ethanol 10.0 Triethanolamine 0.1 antiseptic a very small amount Pigment a very small amount Spices a very small amount Purified water Balance

<Cosmetic formulation example 2. Preparation of nutrient lotion>

As shown in the composition of Table 2 below, a nutrient lotion (lotion, 100 g) containing the flavourzyme treatment of the white spotted radish larva of the present invention was prepared according to a conventional method.

Raw material Content (g) Flavourzyme treatment of white-spotted radish larva 0.1 Sitosterol 1.7 Polyglyceryl 2-oleate 1.5 Ceteares 1.2 cholesterol 1.5 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Sunflower Oil 10.0 Carboxyvinyl polymer 0.2 Xanthan gum 0.3 antiseptic a very small amount Spices a very small amount Purified water Balance

Claims (8)

Flavorzyme (flavourzyme)-treated white spotted flower radish ( Protaetia brevitarsis seulensis) larvae of the enzyme treatment, characterized in that it contains the anti-inflammatory composition. The method of claim 1,
The enzyme-treated product of the white spotted radish larvae is a low molecular weight of 3 kDa or less obtained by filtering the liquid obtained by treating the powder obtained by drying the white spotted radish larva with flavozyme using an ultrafiltration membrane (centrifugal filter). Anti-inflammatory composition, characterized in that the protein concentrate. The method of claim 1,
The enzyme-treated product of the white spotted radish larva is one from the group consisting of NO (nitric oxide), iNOS (inducible nitric oxide synthase), TNF-α (tumor necrosis factor-α), and IL-1β (interleukin-1β). Anti-inflammatory composition, characterized in that to suppress the production of the above-selected inflammation-related factors. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that it contains the composition of claim 1. The method of claim 4,
The inflammatory diseases include inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, cystitis, nephritis and neuritis. Pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that selected from the group consisting of. Health functional food for preventing or improving inflammatory disease, characterized in that it contains the composition of claim 1. The method of claim 6,
The inflammatory diseases include inflammatory bowel disease, inflammatory collagen vascular disease, glomerulonephritis, inflammatory skin disease, sarcoidosis, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, sore throat, bronchitis, pneumonia, pancreatitis, sepsis, cystitis, nephritis and neuritis. Health functional food for preventing or improving inflammatory diseases, characterized in that selected from the group consisting of. An anti-inflammatory cosmetic composition comprising the composition of claim 1.

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