KR20220133351A - Method for producing black radish tea and composition for anti-inflammation using extract of black radish tea - Google Patents

Abstract

The present invention relates to a producing method of a black radish tea and a composition for anti-inflammatory using an extract of the black radish tea made of: a black radish washing step of washing black radish; a drying step of drying after cutting the black radish washed through the black radish washing step; and a roasting step of roasting the dried black radish through the drying step. The present invention not only enable the black radish tea produced through the process to have excellent taste and aroma, but also shows an effect of enabling to be easily ingested as an active ingredient showing anti-inflammatory effect contained in the black radish in a tea form.

Description

Method for producing black radish tea and composition for anti-inflammatory using extract of black radish tea {METHOD FOR PRODUCING BLACK RADISH TEA AND COMPOSITION FOR ANTI-INFLAMMATION USING EXTRACT OF BLACK RADISH TEA}

The present invention relates to a method for producing black radish tea and an anti-inflammatory composition using an extract of the black radish tea. It relates to a method for producing black radish tea that can be easily ingested in the form and to an anti-inflammatory composition using the extract of black radish tea.

Radish (Raphanus sativus) is a root vegetable grown and consumed all over the world, and in some countries it is an important ingredient that is considered part of the table. I prefer to grind it and drink it in the form of juice to pursue it.

The skin color of radish varies, such as red, purple, black, yellow, white and pink, but the flesh is usually white, and the roots of radishes vary greatly in taste, size and length.

Radishes are also used as home remedies to treat various ailments such as jaundice, gallstones, liver disease, rectal prolapse, indigestion and other stomach pains. Included. It also contains various water-soluble vitamins (B ₁ , B ₂ , B ₃ , B ₅ , B ₆ , B ₉ , C) and minerals (calcium, iron, magnesium, manganese zinc, potassium and phosphorus). Among other things, radishes have recently been found to contain unique bioactive compounds with potential health benefits for humans, including glucosinolates (eg, glucoraphanin, 4-hydroxyglucobrasicin). , glucoerucine, glucorafasatin, glucobrasicin, 4-methoxyglucobracicin, neoglucobrasicin), carbinol and isothiocyanates (eg, sulforafen, sulforaphane), and the like.

Black radish (Raphanus sativus L. Var), a type of radish, is a cruciferous vegetable that contains high glucosinolate concentrations as well as sulfate and cysteine-rich proteins that are considered precursors for GSH synthesis. As a therapy, it is used to treat flatulence, gallstones, asthma, bronchitis and other respiratory diseases.

On the other hand, inflammation is a defense reaction of the living body against infection from external infectious agents such as external physical and chemical stimuli, bacteria, fungi, viruses, and various allergens, and is a part of the innate immune response. It begins with macrophages recognizing and attacking non-self through patterns on the cell surface that are specific to pathogens. During the inflammatory reaction, plasma is accumulated in the inflammatory site to dilute the toxicity secreted by the bacteria, blood flow increases, and symptoms such as erythema, pain, edema and fever are accompanied.

Although various biochemical phenomena are involved in the inflammatory response, macrophages are known to play an important role in the inflammatory response by generating nitric oxide (NO) and various inflammatory cytokines by chemical stimulation. Nitric oxide is synthesized from L-arginine by the action of nitric oxide synthase (NOS), and NOS exists in several isoforms. bNOS (brain NOS) present in the brain, nNOS (neuronal NOS) present in the nervous system, and eNOS (endothelial NOS) present in the vascular endothelial system are always expressed at a certain level in the body, and monoxide produced in small amounts by these Nitrogen plays an important role in maintaining homeostasis of the human body by performing various physiological reactions related to blood pressure regulation, neurotransmission, learning and memory. On the other hand, iNOS (induced NOS) whose expression is induced by a certain stimulus produces excessive nitric oxide, and the nitric oxide produced excessively by iNOS reacts with superoxide to form peroxynitrite It acts as a powerful oxidizing agent and damages cells, thereby being involved in various pathological processes, including inflammation and cancer.

Among the functions of COX, the function of PGH synthase is involved in pain and inflammatory responses through the synthesis of PGE ₂ . There are two subtypes of COX, and COX-1 is constitutively expressed in most tissues, whereas COX-2 is rapidly induced by inflammatory cytokines to play an important role in the inflammatory response.

Bacterial endotoxins such as LPS (lipopolysaccharide) activate NF-κB, a transcription factor, by binding to toll-like receptor 4 (TLR4), and induce the expression of iNOS and COX-2 to induce nitric oxide, inflammatory cytokines, and PGE ₂ It secretes several anti-inflammatory substances.

nitric oxide, Inflammatory cytokines such as TNF-α and IL-6, PGE ₂ , etc. have been reported as important factors inducing inflammatory responses in arthritis, fibromyalgia, and Sjogren's syndrome.

IL-6 is an inflammatory cytokine that plays an important role in the innate immune system, but adult diseases such as cancer, camellia cirrhosis, fatty liver disease, nonalcohol fatty liver disease ), neuropathological diseases, rheumatoid arthritis (RA), and autoimmune disorders. When an inflammatory signal is received from macrophages, such as monocytes, kuuper cells, and microglia, TNF-a, IL6, and IL1b are secreted mainly from liver tissue and NFkB (nuclear factor) It is activated by κ-light-chain-enhancer of activated B cells) and STAT3 (Signal transducer and activator of transcription 3) and activates inflammation by promoting COX-2 and iNOS.

Therefore, inhibitors such as nitric oxide and inflammatory cytokines, iNOS inhibitors, and COX-2 inhibitors can be used as therapeutic agents for inflammatory diseases.

Republic of Korea Patent Registration No. 10-2002284 (2019.07.16) Republic of Korea Patent Registration No. 10-2083472 (2020.02.25)

It is an object of the present invention to provide a method for manufacturing black radish tea, which not only has excellent taste and fragrance, but also enables simple intake of active ingredients exhibiting anti-inflammatory effects contained in black radish in the form of tea, and anti-inflammatory using the extract of black radish tea To provide a composition for

Another object of the present invention is to provide a method for producing black radish tea, which exhibits excellent anti-inflammatory effect by improving the dissolution effect of active ingredients exhibiting anti-inflammatory effects contained in black radish, and an anti-inflammatory composition using the extract of black radish tea. .

An object of the present invention is a black radish washing step of washing black radish, a drying step of cutting and drying the black radish washed through the black radish washing step, and a roasting step of roasting the dried black radish through the drying step, characterized in that It is achieved by providing a method for producing black radish tea.

According to a preferred feature of the present invention, in the drying step, the black radish washed through the black radish washing step is cut and frozen at a temperature of -30 to -20°C, and then frozen at a temperature of 30 to 40°C for 30 to 40 hours. It is supposed to be dried.

According to a more preferred feature of the present invention, it is assumed that the rattling is repeated 2 to 3 times.

According to a more preferred feature of the present invention, the roasting is performed at a temperature of 100 to 120° C. for 4 to 6 minutes.

In addition, the object of the present invention can also be achieved by providing an anti-inflammatory composition comprising an extract of black radish tea prepared by the method for producing black radish tea as an active ingredient.

According to a preferred feature of the present invention, as for the extract of black radish tea, the solid extract obtained by extracting black radish tea with methanol is dissolved in distilled water, adsorbed on adsorption resin HP-20, and eluted with distilled water, and the mass concentration is 45 It is assumed that it is prepared by eluting with ethanol of 55% to 55%.

According to a more preferred feature of the present invention, the solid extract is obtained by immersing the black radish tea in methanol having a mass concentration of 75 to 85% for 20 to 30 hours, and after irradiating ultrasonic waves for 40 to 80 minutes, the methanol It is assumed to be manufactured by removing it.

The method for producing black radish tea according to the present invention and the anti-inflammatory composition using the extract of the black radish tea not only have excellent taste and fragrance, but also the active ingredient that exhibits the anti-inflammatory effect contained in the black radish can be easily ingested in the form of tea. It exhibits an excellent effect to

In addition, it has an excellent effect of providing an anti-inflammatory composition using black radish tea and an extract of black radish tea, which exhibits an excellent anti-inflammatory effect by improving the dissolution effect of the active ingredient having an anti-inflammatory effect contained in black radish.

1 is a flowchart illustrating a method for manufacturing black radish tea according to the present invention.
Figure 2 is a result showing the cell viability in the LPS-stimulated mouse macrophage cell line (RAW 264.7 cells) of the black radish tea extract prepared in Example 1 (baked) and Comparative Example 1 (baked) of the present invention.
3 shows the activity of the black radish tea extract prepared in Example 1 (baked) and Comparative Example 1 (baked) of the present invention to inhibit the production of nitric oxide in LPS-stimulated mouse macrophages (RAW 264.7 cells); This is a result indicating whether or not cytotoxicity is present.
FIG. 4 shows mRNA expression of iNOS, an inflammatory factor, in mouse macrophage cell lines (RAW 264.7 cells) stimulated with LPS in the black radish tea extract prepared in Example 1 (black radish) and Comparative Example 1 (black radish) of the present invention. It is a result showing the activity of inhibiting.
FIG. 5 shows COX-2, an inflammatory factor, in a mouse macrophage cell line (RAW 264.7 cells) in which the black radish tea extract prepared in Example 1 (black radish) and Comparative Example 1 (black radish) of the present invention was stimulated with LPS. Results showing the activity of inhibiting mRNA expression.
6 is a result showing the activity of the black radish tea extract prepared in Example 1 (black radish) and Comparative Example 1 (black radish) of the present invention to inhibit the expression of IL-1b, an inflammatory cytokine.
7 shows that the black radish tea extract prepared in Example 1 (black radish) and Comparative Example 1 (black radish) of the present invention inhibits IL-6 expression in LPS-stimulated mouse macrophages (RAW 264.7 cells). It is a result showing the activity.
8 is a graph showing that the black radish tea extract prepared in Example 1 (black radish) and Comparative Example 1 (black radish) of the present invention inhibits mRNA expression of TNFα in LPS-stimulated mouse macrophages (RAW 264.7 cells) This is the result of activity.
9 shows the activity of inhibiting iNOS protein expression in LPS-stimulated mouse macrophage cell lines (RAW 264.7 cells) of black radish tea extract prepared in Example 1 (baked) and Comparative Example 1 (baked) of the present invention; is the result
10 shows the activity of the black radish tea extract prepared in Example 1 (baked) and Comparative Example 1 (baked) of the present invention to inhibit COX-2 protein expression in LPS-stimulated mouse macrophages (RAW 264.7 cells) is the result showing

Hereinafter, a preferred embodiment of the present invention and the physical properties of each component will be described in detail, which is intended to describe in detail enough that a person of ordinary skill in the art to which the present invention pertains can easily carry out the invention, This does not mean that the technical spirit and scope of the present invention is limited.

The method for producing black radish tea according to the present invention includes a black radish washing step (S101) for washing black radish, a drying step (S103) of cutting and drying the black radish washed through the black radish washing step (S101), and the drying step (S103) ) through the roasting step (S105) of roasting the dried black radish.

The black radish washing step (S101) is a step of washing black radish, and consists of washing the black radish with purified water to remove soil and foreign substances remaining on the surface of the black radish, and removing the fine roots of the black radish.

At this time, the black radish is not particularly limited, but it is preferable to use black radish for wintering from Jeju harvested in August to December 2020 in Seongsan-eup, Seogwipo-si, Jeju-do.

The drying step (S103) is a step of drying after cutting the black radish washed through the black radish washing step (S101). After cutting to a thickness, it is naturally dried at a temperature of 1 to 10° C. in a shaded place, or it consists of a process of freeze-drying.

Black radish dried through the above process has a reduced moisture content and thus not only improves preservation, but also improves dissolution efficiency of active ingredients contained in black radish.

At this time, the freeze-drying is performed by cutting the black radish washed through the black radish washing step, freezing it at a temperature of -30 to -20°C, and then freeze-drying it at a temperature of 30 to 40°C for 30 to 40 hours. The loss of active ingredients is minimized in black radish frozen at a temperature of

At this time, the freeze-drying step is carried out for 4 hours at a temperature condition of 30° C. and a vacuum degree of 5000 torr, and then proceeds for 26 to 30 hours at a temperature condition of 31 to 40° C. and a vacuum degree of 800 Mtorr, and a temperature condition of 30° C. and 800 Mtorr. It is most preferable to proceed for 4 hours at a vacuum degree of

The roasting step (S105) is a step of roasting the black radish dried through the drying step (S103). When it comes to roasting, meat, medicine, grain, etc. with a little moisture is put in a pot or a pot to prevent burning with strong heat. It refers to the process of roasting and cooking, and is one of the processes used in green tea production.

In the present invention, the roasting step (S105) consists of roasting the black radish dried through the drying step (S103) at a temperature of 100 to 120° C. for 4 to 6 minutes. The roasting process consisting of the above temperature and time More preferably, it is repeated 2-3 times.

In addition, in the case of repeating the roasting process 2 to 3 times as described above, it is preferable to proceed with the roasting process again after the roasted black radish is cooled to room temperature. If repeated, the dissolution efficiency of the active ingredient contained in the dried black radish is further improved.

After the above roasting step (S105), the production of black radish tea according to the present invention is completed, and the manufactured black radish tea can be packaged and commercialized in a tea bag, etc. It can be consumed easily by brewing it for about 10 minutes.

If the temperature of the roasting step (S105) is less than 100°C, the effect of improving the dissolution efficiency of the active ingredients contained in the dried black radish through the roasting process is insignificant, and the temperature of the roasting step (S105) exceeds 120°C. This is undesirable because the effective ingredients or nutrients contained in the black radish are destroyed without significantly improving the above effects.

In addition, the black radish tea prepared through the above process is put into a tea bag as described above and after it is commercialized, brewed in hot water to drink, and a solid extract obtained by extracting black radish tea with methanol is dissolved in distilled water and adsorbed After adsorption to the resin HP-20, it is eluted with distilled water and eluted with ethanol having a mass concentration of 45 to 55% to prepare an anti-inflammatory composition.

The solid extract is preferably prepared by immersing the black radish tea in methanol having a mass concentration of 75 to 85% for 20 to 30 hours, irradiating ultrasonic waves for 40 to 80 minutes, and then removing the methanol.

In this case, the "inflammation" is specified as a local or systemic biodefense reaction against an external physical or chemical stimulus or infection or autoimmunity from external infectious agents such as bacteria, fungi, viruses, and various allergens. Activation of inflammatory mediators and enzymes related to immune cells (eg, iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, secretion of NO, TNF-α, IL-6, etc.), infiltration of body fluids, cell migration, tissue destruction It is accompanied by a series of complex physiological reactions such as erythema, pain, edema, fever, and is externally manifested by symptoms such as decrease or loss of specific body functions, etc. The inflammatory disease is acute, chronic, ulcerative, or allergic. or necrotic, whether any disease is acute, chronic, ulcerative, allergic or necrotic, as long as it falls within the definition of inflammatory disease as described above. 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, migraine, headache, back pain, fibromyalgia, myofascial disease, viral infection (such as hepatitis C infection), bacterial infection, fungal infection, burn, surgical or dental wound, 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.

In addition, the anti-inflammatory composition according to the present invention may contain the active ingredient in any amount (effective amount) as long as it can exhibit the inflammatory disease improvement activity intended for treatment, etc. according to the use, formulation, purpose of formulation, etc. A typical effective amount will be determined within the range of 0.001% to 15% by weight, based on the total weight of the composition. As used herein, the term "effective amount" refers to the intended medical and pharmacological effects, such as improvement of inflammatory diseases, when the composition of the present invention is administered to a mammal, preferably a human, to which it is applied during the administration period as suggested by a medical professional. It refers to the amount of the active ingredient included in the composition of the present invention that can be used.The effective amount can be determined empirically within the scope of ordinary skill in the art.

In addition, the anti-inflammatory composition of the present invention is administered through addition of similar activities such as anti-allergic activity, skin protection activity (suppression of skin damage caused by UV rays, skin moisturizing, etc.), in addition to the active ingredient, for increasing and reinforcing the anti-inflammatory effect However, in order to enhance the convenience of ingestion, any compound or natural extract that has already been verified for safety in the art and known to have the corresponding activity may be further included.

In addition, the anti-inflammatory composition of the present invention can be applied to a food composition, a pharmaceutical composition, a cosmetic composition, etc., the food composition can be prepared in any form, for example, beverages such as tea, juice, carbonated beverage, ionic beverage, Processed oils such as milk and yogurt, gums, rice cakes, Korean confectionery, bread, confectionery, noodles, etc., health such as tablets, capsules, pills, granules, liquids, powders, pieces, pastes, syrups, gels, jellies, bars, etc. It may be prepared as functional food formulations, etc.

In addition, when applied as a pharmaceutical composition, it may be prepared as an oral dosage form or a parenteral dosage form according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of an active ingredient and does not have toxicity beyond what the application (prescription) target can adapt.

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers according to methods known in the art together with suitable carriers It can be prepared in a formulation such as When the pharmaceutical composition of the present invention is prepared for parenteral use, it may be formulated in the form of injections, transdermal administrations, nasal inhalants and suppositories together with suitable carriers according to methods known in the art. In the case of formulation for injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterilization for injection. Water, an isotonic solution such as 5% dextrose, etc. can be used. When formulated for transdermal administration, it may be formulated in the form of an ointment, a cream, a lotion, a gel, an external solution, a pasta agent, a liniment agent, an air roll, and the like. In the case of nasal inhalants, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and the like. witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like may be used.

In addition, when the anti-inflammatory composition according to the present invention is applied as a cosmetic composition, its use may be understood as alleviation of inflammatory skin irritation. The cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the active ingredient, for example, conventional adjuvants such as stabilizers, solubilizers, surfactants, vitamins, pigments and fragrances, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

Hereinafter, the method for producing black radish tea according to the present invention and the physical properties of the anti-inflammatory composition using the black radish tea extract prepared through the production method will be described with reference to examples.

<Production Example 1> Preparation of black radish tea

In Seongsan-eup, Seogwipo-si, Jeju Special Self-Governing Province, the roots of Jeju black radish harvested in October 2020 were washed with fresh water three times or more, dried naturally for 72 hours at a temperature of 5℃ in a shaded place, Black radish tea was prepared by cutting to thickness, roasting the cut black radish at a temperature of 110° C. for 5 minutes, cooling it to room temperature, and repeating the process of roasting for the above temperature and time again twice.

<Example 1>

300 g of black radish tea prepared in Preparation Example 1 was immersed in 6L of 80% methanol for 24 hours, irradiated with ultrasonic waves for 60 minutes, the supernatant was taken, and the remaining residue was extracted in the same manner as above, and then mixed with filter paper ( Whatman No 2), the resultant was concentrated with a vacuum rotary concentrator (Heidolph, LABOROTA) at 40° C. and 90 rpm speed to obtain a concentrated solid. Then, it was dissolved again in purified water to a content of 10% (w/w), and freeze-dried to prepare a powdery extract. After dissolving 60 g of this extract in 300 mL of distilled water and filling the HP-20 column, 10 L of distilled water was loaded onto the column, the distilled water was discarded, and 4 L of 50% ethanol was loaded again. The loaded 50% ethanol layer was received in a concentrator, concentrated with a vacuum rotary concentrator (Heidolph, LABOROTA) at a speed of 90 rpm, and then the concentrated solid was dissolved in purified water to a content of 10% (w/w), and freeze-dried to form a powder An anti-inflammatory composition was prepared.

<Comparative Example 1>

Proceed in the same manner as in Example 1, except that the black radish roots from Jeju harvested in February 2021 in Seongsan-eup, Seogwipo-si, Jeju-do were washed with fresh water 3 times or more, dried in cold air, and then pulverized to use ground black radish. To prepare an anti-inflammatory composition.

The anti-inflammatory activity of the anti-inflammatory composition prepared in Example 1 and Comparative Example 1 was measured and shown in FIGS. 2 to 9 below.

Anti-inflammatory activity was obtained by purchasing a murine macrophage cell line RAW 264.7 from the American Type Culture Collection (ATCC, Rockville, MD, USA), 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin. (100 μg/mL) and stored in Dulbeccos Modified Eagles Medium (DMEM; GIBCO Inc.). These cells were cultured in subconfluence under conditions of 95% air, 5% CO ₂ humidified air at 37° C., and subcultured every 3 days.

The degree of nitric oxide production was measured by using the nitric oxide assay method using cultured RAW 264.7 cells. Specifically, the concentration was adjusted to 1×10 ⁵ cells/mL using DMEM medium supplemented with 10% FBS, and then inoculated into a 24-well plate, and samples of each concentration and LPS (1㎍/mL) were simultaneously administered treated and cultured for 24 hours. The amount of nitric oxide produced was measured using Griess reagent 1% (w/v) (sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid) to determine the nitric oxide present in the cell culture medium. It was measured in the form of ions (NO ² -). 100 μl of cell culture supernatant and 100 μl of grease reagent were mixed and reacted for 10 minutes in a 96-well plate, and then absorbance was measured at 540 nm. The amount of nitric oxide produced was quantified by comparing sodium nitrite (NaNO ₂ ) as a standard.

Cytotoxicity was measured by lactate dehydrogenase (LDH) assay. Specifically, RAW 264.7 cells (1.5 cells/mL) were simultaneously treated with the samples of each concentration and LPS (1 μg/mL) in DMEM medium and cultured for 24 hours. A culture medium was obtained and centrifuged at 3,000 rpm for 5 minutes. LDH (lactate dehydrogenase) assay was measured using a non-radioactive cytotoxicity assay kit (Promega), and 50 μl of culture medium obtained by centrifugation and 50 μl of reconstituted substrate mix were added to a 96-well plate, and reacted at room temperature for 30 minutes. After adding 50 μl of stop solution, absorbance was measured at 490 nm using a microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA). The average absorbance value for each sample group was obtained, and cytotoxicity was evaluated by comparing it with the absorbance value of the control group (LDH control, 1:5000).

To evaluate the inhibitory efficacy of Prostaglandin E2 (PGE ₂ ) production, RAW 264.7 cells were adjusted to 1×10 ⁵ cells/mL using DMEM medium, inoculated in a 24 well plate, and cultured 18 hours before in a 5% CO ₂ incubator, Thereafter, the medium was removed, and the sample of each concentration and a new medium containing LPS (1 μg/mL) were treated at the same time and cultured under the same conditions as the previous culture. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 min) to measure PGE ₂ to obtain a supernatant. The measurement of PGE ₂ was quantified using a PGE ELISA kit (R&D Systems Inc., Minneapolis, MN, USA), and the r ² value of the standard curve for the standard was 0.99 or more.

To evaluate the inhibitory efficacy of IL-6 production, RAW 264.7 cells (1×10 ⁵ cells/mL) were inoculated in a 6-well plate using a medium without FBS and cultured for 14 hours. After one hour of treatment with the extract, IL-6 (10 ng/ml) was treated for 30 minutes, and cells were collected.

To evaluate the inhibitory efficacy of inflammatory cytokines (TNF-α, IL-1β and IL-6) production, RAW 264.7 cells (1×10 ⁵ cells/mL) were inoculated into a 24-well plate after adjusting the number of cells using DMEM medium. and incubated for 18 hours in a 5% CO ₂ incubator. Thereafter, the medium was removed, and RAW 264.7 cells were treated with a new medium containing 50 μl of the sample of each concentration and 450 μl of LPS (1 μg/mL) at the same time, and cultured under the same conditions as the pre-culture. After 24 hours, the culture medium was centrifuged (12,000 rpm, 3 minutes) to measure the content of pro-inflammatory cytokines in the supernatant obtained. All samples were stored at -20°C or lower until quantification. The quantification of pro-inflammatory cytokines was quantified using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, MN, USA), and the r ² value of the standard curve for the standard was 0.99 or more.

For the evaluation of the inhibitory efficacy of INOS and COX-2 expression (western-blotting), the cultured cells are collected and washed with PBS (phosphate buffered saline) 2-3 times, followed by a cell lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM dithiothreitol, 1 mM phenyl methyl sulfonyl fluoride, 25 ug/mL aprotinin, 25 ug/mL leupeptin] was added. After dissolving at 4°C for 30 minutes, centrifugation was performed at 4°C and 15,000 rpm for 15 minutes to remove cell membrane components. Protein concentration was standardized with BSA (bovine serum albumin) and quantified according to the manufacturer's protocol using Bio-Rad Protein Assay Kit (Bio-Rad). Specifically, 20-30 μg of the isolated protein was denatured and separated by 8-12% mini gel SDS-PAGE, and it was transferred to a PVDF (polyvinylidene difluoride) membrane (BIO-RAD, Richmond, CA, USA) at 200 mA for 2 hours. It was transferred. And the blocking of the membrane (membrane) was performed for 2 hours at room temperature in a TTBS (0.1% Tween 20 + TBS) solution containing 5% skim milk. Anti-mouse iNOS (Calbiochem, La Jolla, CA, USA) as an antibody to examine the expression level of iNOS is anti-mouse COX-2 (BD Biosciences Pharmingen, San Jose, CA, USA) was diluted 1:1000 in TTBS solution, reacted at room temperature for 2 hours, and then washed 3 times with TTBS. As a secondary antibody, HRP (horse radish peroxidase)-conjugated anti-mouse IgG (Amersham Pharmacia Biotech, Little Chalfont, UK) was diluted 1:5000 and reacted at room temperature for 30 minutes, followed by washing 3 times with TTBS. After reaction with ECL substrate (Amersham Biosciences, Piscataway, NJ, USA) for 1 to 3 minutes, it was exposed to X-ray film.

qPCR showed IL-1, IL-6, TNF-α, COX-2, and iNOS (Table 1). To perform RT-PCR, total RNA was isolated from RAW 264.7 cells using NucleoSpin RNA reagent (Macherey-Nagel, Germany) according to the manufacturer's manual. Using the isolated total RNA as a template, cDNA was synthesized using the First Strand cDNA Synthesis Kit (Toyobo, Japan) according to the manufacturer's manual. For qPCR, the synthesized cDNA was used as a template and a gene-specific oligo primer was mixed with a premix (5Х HOT FIREPol EvaGreen qPCR Supermix; Solids BioDyne, Estonia), followed by 35 cycles in a Bioer thermal cycler (Hangzhou, China). The primers used for PCR are shown in Table 1 below, and the primers were custom-made by Bioneer (Korea).

<Table 1> Primer sequences used in real-time quantitative polymerase chain reaction analysis

In Figure 2 below, MTT is the result of performing MTT assay 24 hours after processing the extracts of Example 1 (baked) and Comparative Example 1 (baked) of the present invention to RAW264.7 cells, which are macrophage monocy, for each concentration. indicated. As a result, it showed lower cell viablity at night than the control, but showed the same cell viability as the LPS-treated group.

In addition, the results for the nitric oxide production inhibitory activity and cytotoxicity are shown in Figure 3 below. Referring to Figure 3 below, the anti-inflammatory composition of Example 1 (Neam) inhibits nitric oxide production in a concentration-dependent manner, It can be seen that there is no particular cytotoxicity. However, the anti-inflammatory composition of Comparative Example 1 (bean) exhibited an inhibitory effect on the production of nitric oxide only at a high concentration, and did not exhibit an inhibitory effect at a low concentration.

In addition, the results for the inhibitory activity of iNOS and COX-2 production are shown in FIGS. 4 to 5 below. It was shown that the anti-inflammatory composition of Example 1 (black radish) inhibited the production of these enzymes in a concentration-dependent manner.

However, in the case of COX-2, both of the anti-inflammatory compositions of Example 1 (black radish) and Comparative Example 1 (black radish) showed a tendency to inhibit at high concentrations, but were not inhibited at low concentrations. Therefore, it can be seen that black radish tea extract inhibits COX-2 only at high concentrations.

In addition, the results for the inhibitory activity of the inflammatory cytokine IL-1β are shown in Figure 6 below, and it can be seen that the expression of the inflammatory cytokine IL-1β is inhibited only in the anti-inflammatory composition of Example 1 (black red). . It can be seen that the high concentration of the anti-inflammatory composition of Comparative Example 1 (black radish) promotes the expression of IL-1β.

In addition, the results for the inhibitory activity of the inflammatory cytokines IL-6 and TNF-a are shown in FIGS. 7 to 8 below. Expression of the inflammatory cytokine IL-1β only in the anti-inflammatory composition of Example 1 (black red) It can be seen that this is suppressed.

In addition, the results for the iNOS and COX-2 production inhibitory activity are shown in FIGS. 9 to 10 below. The anti-inflammatory composition of Example 1 (baked) inhibits the protein expression of iNOS and COX-2 enzymes in a concentration-dependent manner. it can be seen that

However, it was found that the anti-inflammatory composition of Comparative Example 1 (Ninja) showed a tendency to inhibit INOS and COX-2 at a high concentration, but did not inhibit it at a low concentration. Therefore, it can be seen that black radish tea extract inhibits COX-2 at high concentrations.

Therefore, the method for producing black radish tea according to the present invention and the anti-inflammatory composition using the extract of black radish tea not only have excellent taste and fragrance, but also conveniently consume the active ingredient that exhibits the anti-inflammatory effect contained in black radish in the form of tea. can do.

In particular, the extract of black radish tea according to the present invention improves the dissolution effect of the active ingredient exhibiting the anti-inflammatory effect contained in the black radish to suppress nitric oxide production, inhibit INOS mRNA expression, and inflammatory cytokines IL-1b, IL-6, TNF -a, mRNA expression suppression, and significantly suppresses the expression of INOS and COX-2 protein, thereby exhibiting an excellent anti-inflammatory effect.

Although the present invention has been described in detail with reference to the embodiments, it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical spirit of the present invention, and it is natural that such variations and modifications belong to the appended claims.

S101; Black radish washing step
S103; drying stage
S105 ; Steps

Claims (7)

a black radish washing step of washing the black radish;
a drying step of cutting and drying the black radish washed through the black radish washing step; and
A method for producing black radish tea, comprising: a roasting step of roasting the dried black radish through the drying step.
The method according to claim 1,
In the drying step, the black radish washed through the black radish washing step is cut, frozen at a temperature of -30 to -20°C, and then freeze-dried at a temperature of 30 to 40°C for 30 to 40 hours. A method for manufacturing tea-free tea.
The method according to claim 1,
The method for producing black radish tea, characterized in that the roasting is repeated 2-3 times.
The method according to claim 1,
The roasting is a method of producing black radish tea, characterized in that it is made for 4 to 6 minutes at a temperature of 100 to 120 ℃.
An anti-inflammatory composition comprising, as an active ingredient, an extract of black radish tea prepared by the method for producing black radish tea according to any one of claims 1 to 4.
6. The method of claim 5,
The extract of black radish tea is obtained by dissolving a solid extract obtained by extracting black radish tea with methanol, adsorbing it on an adsorption resin HP-20, eluting with distilled water, and eluting with ethanol having a mass concentration of 45 to 55%. An anti-inflammatory composition, characterized in that it is prepared.
7. The method of claim 6,
The solid extract is characterized in that it is prepared by immersing the black radish tea in methanol having a mass concentration of 75 to 85% for 20 to 30 hours, irradiating ultrasonic waves for 40 to 80 minutes, and then removing the methanol. composition for inflammation.

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