KR102058022B1 - Composition for antioxidant and anti-inflammatory comprising fraction of Ledum palustre L. extract as effective component - Google Patents

Abstract

The invention Baishan tea (Ledum palustre L.) Antioxidant and anti-inflammatory composition containing ethyl acetate fraction of ethanol extract as an active ingredient, and may be useful as an anti-inflammatory and anti-inflammatory food and cosmetic composition and a pharmaceutical composition for the prevention and treatment of inflammatory diseases. have.

Description

Composition for antioxidant and anti-inflammatory comprising fraction of Ledum palustre L. extract as effective component

White acid car of the present invention palustre L.) The ethyl acetate fraction of ethanol extracts inhibits the production of antioxidant and inflammatory mediators NO and inflammatory cytokines, and is an anti-inflammatory and anti-inflammatory food and cosmetic composition and a pharmaceutical composition for the prevention and treatment of inflammatory diseases. It can be usefully used.

With the development of industrial society and the aging population, the desire to lead a healthy life is increasing, and consumers' interest in functional foods and cosmetics is increasing all over the world. Therefore, research and development has been actively conducted, and evidence-based studies for active ingredient discovery such as antioxidant and anti-inflammatory effects are actively being conducted. Most of the active oxygen are unstable and have the property of losing or gaining electrons to go to a more stable state, and their high reactivity causes oxidative damage in vivo. The free radicals produced during the oxidation process in the body are removed by antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase in the body, but ROS (reactive oxygen species) is lipid peroxidation, It causes damage such as protein oxidation, protease activation, and DNA oxidation. As a result of this damage, inflammatory diseases such as hypertension, diabetes, atopic dermatitis, and rheumatoid arthritis develop. As the oxidative stress caused by excessive ROS is known to be reduced by antioxidants, studies to develop antioxidants are continuously conducted. As the side effects of synthetic antioxidants increase, Research is ongoing. Plant-derived secondary metabolites are known to prevent cell damage due to oxidation by inhibiting or eliminating the production of free radicals and free radicals. DPPH (1,1-diphenyl-) is a method of confirming radical scavenging ability from natural products. 2-picrylhydrazyl) and ABTS (2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) analyzes are widely studied. Inflammatory reactions identify and eliminate invasion of various foreign substances such as external pathogens into the human body. It is a primary immune response, a self-defense system that maintains homeostasis, and macrophages are involved in the production of nitric oxide (NO), a representative inflammatory mediator. It is excessively produced by the virus and intensifies inflammation.TNF-α (tumor necrosis factor-α) and IL-1β are caused by endotoxin LPS (lipopolysaccharide) present in the extracellular membrane of Gram-negative bacteria in macrophages. increase the secretion of cytokines such as (interleukin-1β) and IL-6 (interleukin-6), etc. In addition, NO produced by iNOS and PGE2 (prostaglandin E2) produced by COX-2 (cyclooxygenase-2) Inflammatory factors are also produced, which in turn stimulate the production of NO and ROS, resulting in excessive inflammatory reactions, which can cause chronic inflammatory diseases, thus preventing and treating various chronic inflammatory diseases. For this purpose, the development of functional materials with antioxidant and anti-inflammatory effects is important.

Baeksan tea ( Ledum palustre L.) is an evergreen subfamily of azaleas growing in Mt. Baekdu Mountain. It grows under the forests of high mountains. It has young shoots from the roots and dense brown hairs. In Siberia and Northern Europe, it is often used in homeopathy. In Korea, it is refreshed after a meal by drinking tea. Baeksan tea is also known to be used as a preservative and leaves are used for medicinal purposes. It contains tannin and terpene in the leaves and roots of white tea and is effective in chronic bronchitis, menstrual irregularities, infertility, gastric ulcer and rheumatoid arthritis. However, there are many studies on white tea, and the antioxidant and anti-inflammatory activity of white tea has not been reported.

Korean Patent No. 1456287 discloses a method for preparing antioxidant and anti-inflammatory functional tea using Pear cancer tea leaves, and Korean Patent Application Publication No. 2015-0071698 discloses a method for preparing antioxidant and anti-inflammatory functional oak sequence. It is different from the composition for antioxidant and anti-inflammatory, containing the fraction of Baeksan tea extract of the present invention as an active ingredient.

The present invention is derived from the above requirements, in the present invention, in extracting white acid tea, the antioxidant and anti-inflammatory activity of the extracting solvent and the solvent fraction was verified, thereby improving the antioxidant and anti-inflammatory activity in the extract. The purpose is to establish a method of producing white acid tea extract.

In order to achieve the above object, the present invention provides an antioxidant and anti-inflammatory health functional food containing ethyl acetate fraction of Baeksan tea ethanol extract as an active ingredient.

The present invention also provides an antioxidant and anti-inflammatory cosmetic composition containing ethyl acetate fraction of baeksan tea ethanol extract as an active ingredient.

The present invention also provides an anti-inflammatory pharmaceutical composition containing ethyl acetate fraction of baeksan tea ethanol extract as an active ingredient.

The fraction of Baeksan tea extract of the present invention can be usefully used as an anti-inflammatory and anti-inflammatory food and cosmetic composition and a pharmaceutical composition for the prevention and treatment of inflammatory diseases. And as an anti-inflammatory functional material.

1 is a graph comparing the cytotoxicity of white tea extract and fractions.
Figure 2 is a graph comparing the NO production amount of white acid tea extract and fractions.
Figure 3 is a graph comparing the production amount of prostaglandin E2 of white tea extract and fractions.
Figure 4 is a graph comparing the amount of inflammatory cytokine production of white tea extract and fractions.

According to an aspect of the invention there is provided Baishan tea (Ledum palustre L.) Provides an antioxidant and anti-inflammatory health functional food containing ethyl acetate fraction of ethanol extract as an active ingredient.

In the dietary supplement according to an embodiment of the present invention, the baeksan tea ethanol extract is preferably 1 to 20-28 hours after adding 18-22 times (v / w) 60-80% ethanol to Baeksan tea. It may be an extract extracted three times, more preferably 70% ethanol 20 times (v / w) added to the white tea may be an extract extracted twice for 24 hours.

The antioxidant and anti-inflammatory health functional food of the present invention may be prepared in any one of a powder, granules, pills, tablets, capsules, candy, syrups and beverages, but is not limited thereto.

When the health functional food of the present invention is used as a food additive, the health functional food may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be appropriately used depending on the purpose of use (prevention or improvement). In general, the health functional food of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw material in the production of food or beverage.

However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of dietary supplement. Examples of foods to which the health functional food can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea drinks Alcoholic beverages and vitamin complexes, and includes all of the health foods in the conventional sense.

In addition, the health functional food of the present invention can be prepared as food, in particular functional food. Functional foods of the present invention include ingredients that are commonly added in food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates can be monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g. dextrins, cyclodextrins, etc.) or sugar alcohols (e.g. , Xylitol, sorbitol, erythritol and the like). The flavourant may be a natural flavourant (eg, taumartin, stevia extract, etc.) and a synthetic flavourant (eg, saccharin, aspartame, etc.).

Various nutritional supplements, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated beverages Carbonation agent etc. which are used for may be contained further. The ratio of the above added ingredients is not critical, but is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food of the present invention.

The present invention is also a white acid ( Ledum palustre L.) Provides an anti-inflammatory and anti-inflammatory cosmetic composition containing ethyl acetate fraction of ethanol extract as an active ingredient.

The cosmetic composition of the present invention is any one selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. It may have one formulation, but is not limited thereto. The cosmetic composition consisting of each of these formulations may contain various bases and additives necessary for the formulation of the formulation and are suitable, and the type and amount of these components can be easily selected by those skilled in the art.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, the carrier component is animal fiber, vegetable fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. And the like can be used.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component, and in particular, in the case of a spray, additionally chlorofluorohydro Propellants such as carbon, propane-butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. Fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, acethionate, an imidazolinium derivative, methyl taurate, and sarcosinate Fatty acid amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention may further contain an excipient including a fluorescent substance, a fungicide, a caustic agent, a moisturizer, a fragrance, a fragrance carrier, a protein, a solubilizer, a sugar derivative, a sunscreen agent, a vitamin, a plant extract, and the like. .

The present invention is also a white acid ( Ledum palustre L.) It provides an anti-inflammatory pharmaceutical composition containing the ethyl acetate fraction of ethanol extract as an active ingredient.

The pharmaceutical compositions of the present invention may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may comprise at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycero gelatin and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and during parenteral administration, it is preferable to select an external skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection method. It is not limited.

The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The dosage of the pharmaceutical composition of the present invention may be used in a variety of ranges depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

1. Materials and Methods

(1) Preparation of Experimental Materials and Extracts

Baeksan tea was purchased in May from Pyrogen (Daegu Dalseong-gun) and used in this experiment. After crushing to 40 mesh or less using a grinder (IKA ® Werke GmbH & Co., KG, Staufen, Germany)- It was used for the experiment while storing at 20 ℃. Extraction method was performed by extracting 9 times distilled water to 100 g of crushed hot water, and extracting the sample twice for 3 hours (LPW). Ultrasonification extraction was performed 9 times to 100 g of sample. A glass bottle containing distilled water was repeatedly extracted twice at 40 kHz for 3 hours without touching the bottom of the ultrasonic bath (NXPC-4020P, KODO, Hwaseong, Korea) (LPU). Autoclave extraction was performed by adding 9-fold distilled water to 100 g of a sample ground in an autoclave (JSAC-100, JS Research Inc., Gongju, Korea), and extracting twice at 121 ° C. for 3 hours (LPA). ), 70% ethanol extraction (70% ethanol extraction) was added to 20 g of 70% ethanol 2 L in 100 g of the sample was immersion extraction twice for 24 hours (LPE). Each extract was filtered through filter paper (Whatman No. 2, Maidstone, England) and then concentrated in a rotary vacuum concentrator (WB2000, Heidolph, Schwabach, Germany). The concentrate was freeze-dried (FD SFESM12, Samwon, Busan, Korea) to prepare a powder, the extract powder was stored at -70 ℃ was used for the experiment. Since the obtained extract from the experiment using the most effective 70% ethanol extract was a solvent fraction. 8 g of 70% ethanol extract was suspended in 100 mL of distilled water, followed by 3 times of 1 L of n-hexane (LPE / H), 3 times of 1 L of ethyl acetate (LPE / E). , And sequentially fractionated 3 times (LPE / B) by 1 L of n-butanol (n-butanol), and LPE / H, LPE / E, LPE / E and water layer (LPE / W) were all concentrated to After securing the fractions were lyophilized and used for the experiment while freezing storage at -70 ℃.

(2) DPPH radical scavenging activity

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was determined by the electron donating effect on DPPH. 50 μL of 0.2 mM DPPH solution and 100 μL of the sample were mixed and then reacted at room temperature for 10 minutes, and then the absorbance was measured at 517 nm using a microplate reader (SunriseTM, Tecan Group Ltd., Mannedorf, Switzerland). The DPPH radical scavenging activity rate was calculated from the absorbance ratio of the sample addition group to the sample no addition group.

(3) ABTS radical scavenging activity

ABTS (2,2-azinobis- (3-ethylbenzo-thiazoline-6-sulphonate)) radical scavenging activity was dissolved in potassium persulfate (2.45 mM) in 7 mM ABTS solution to block the light. It was used after reacting for 16 hours. The ABTS solution was adjusted with ethanol so that the absorbance was 0.6-0.8, 100 μL of the solution and 50 μL of the sample were mixed, reacted for 15 minutes at room temperature, and the absorbance was measured at 734 nm using a microplate reader. The ABTS radical scavenging activity rate was calculated from the absorbance ratio of the sample addition group to the sample no addition group.

(4) cell culture

Raw 264.7 cells, a mouse macrophage line, were purchased from the American type culture collection (ATCC, Rockville, MD, USA), and DMEM (dulbecco's) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for cell culture. modified eagle medium, Invitrogen Carlsbad, CA, USA). Raw 264.7 cells were cultured in 75 T-flask at 37 ° C., 5%, CO ₂ incubator conditions.

(5) MTT analysis

In this experiment, cell viability was determined by MTT analysis to determine the effective concentrations of white acid tea extract and solvent fractions on Raw 264.7 cells. The cultured Raw 264.7 cells were inoculated at 5 × 10 ⁴ cells / well in 48 well plates and incubated for 24 hours. After replacing with the same medium, white acid tea extract and fractions were diluted by concentration and incubated in a CO ₂ incubator for 24 hours. 20 μL of 5 mg / mL 3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) solution in cultured cells After treatment, the reaction was carried out for 4 hours. Thereafter, 100 μL of demethyl sulfoxide (DMSO, Duksan Co., Ltd., Ulsan, Korea) was added thereto, stirred for 10 minutes, and the absorbance was measured at 540 nm using a microplate reader.

(6) NO (Nitric oxide) production amount measurement

To measure NO expression, Raw 264.7 cells were seeded in 48 × well plates at 5 × 10 ⁴ cells / well and incubated in a 24 hour CO ₂ incubator. After 24 hours, the cells were replaced with serum free DMEM and treated with LPS (1 μg / mL). After 1 hour, white tea extract and fractions diluted with serum free DMEM were treated by concentration, and then cultured in an 18 hour CO ₂ incubator. The supernatant of the cultured cells and the grease reagent (Sigma) were mixed at a 1: 1 ratio and allowed to react for 10 minutes. The absorbance was then measured at 540 nm using a microplate reader.

(7) PGE2 production amount measurement

Raw 264.7 cells were seeded in 24-well plates to 2 × 10 ⁵ cells / mL and incubated in a 5% CO ₂ incubator for 24 hours. After 24 hours, the cells were exchanged with serum free DMEM medium, treated with LPS 1 μg / mL, pretreated for 1 hour, and then inoculated with white tea extract and fractions. The supernatant of the incubated cells was taken and PGE2 production was measured. PGE2 measurement was quantified using a mouse enzyme-linked immnunosorbent assay (ELISA) kit (R & D system Inc., Minneapolis, MN, USA), and the standard curve R2 value of the standard was 0.99 or more.

(8) Measurement of Inflammatory Cytokine Production

TNF-α, IL-1β and IL-6 production in the cell culture were measured using a mouse enzyme-linked immnunosorbent assay (ELISA) kit. Raw 264.7 cells were inoculated in 6-well plates after adjusting to 5 × 10 ⁵ cells / ml using DMEM medium and incubated in a 5% CO 2 incubator for 24 hours. After treatment with 1 μg / mL LPS cells 1 hour after the white tea extract and fractions were treated by concentration to incubate for 24 hours. The pro-inflammatory cytokine content of the supernatant obtained after incubation was measured, and quantitatively quantified using an ELISA kit, and the standard curve R2 value of the standard was greater than 0.99.

(9) Statistical Processing

All experiments were repeated three times or more, and the average value and standard deviation were expressed. The significance test was performed using Duncan's multiple range test and t-test using SPSS statistics 20 (IBM, Armonk, NY, USA) software package program. .

Example  One. White car  Yield according to extraction method

For each 100 g of dried and crushed white acid tea, the weight of the sample after extraction and freeze drying according to the extract preparation method was 11.62 g, 10.38 g, 8.65 g, 5.82 g in the order of LPA> LPW> LPE> LPU. Yields were 11.62%, 10.38%, 8.65%, 5.82%. As a result of measuring and comparing DPPH and ABTS radical scavenging activity with respect to the four samples obtained above, LPE (freeze-dried sample of 70% ethanol extract) showed 82.3 at a concentration of RC50 = 197.2 μg / mL and 1,000 μg / mL for DPPH radical. It showed% scavenging activity and 99.8% scavenging activity at concentrations of RC50 = 143.5 μg / mL and 1,000 μg / mL for ABTS radicals, showing the best results in both radical scavenging activities (Tables 1 and 3). Accordingly, the solvent fraction was fractionated and freeze-dried according to the fraction preparation method by taking 8 g of LPE, and the weight of each fraction sample obtained was 2.62 g, 1.86 in the order of LPE / W> LPE / B> LPE / E> LPE / H. g, 1.23 g, 0.06 g and the recovery rates were 32.75%, 23.25%, 15.37%, 0,75%.

Example  2. White car  Of extract DPPH Radical  Scavenging activity

DPPH is a water-soluble substance with chemically stabilized free radicals, reduced by ascorbic acid, tocopherol, polyhydroxy aromatics, etc., which results in a deep purple color that is deoxidized from various natural materials. It is widely used to search for substances. Reactive oxygen species (ROS) are mostly eliminated by the body's defense mechanisms, but when they are not removed, they react rapidly with biomolecules, causing protein denaturation, biofilm lipid peroxidation, and DNA damage. Lipid peroxides promoted new radical reactions and caused various diseases. In this experiment, we measured DPPH radical scavenging activity of four solvent fractions against four different extracts and the most effective 70% ethanol extract. DPPH radical scavenging activity of the four extracts according to the extraction method is shown in Table 1. DPPH radical scavenging activity was measured in the order of LPE> LPU> LPA> LPW at a concentration of 1,000 μg / mL, and the DPPH radical scavenging activity of the solvent fraction from the LPE showing the highest activity was determined as LPE / mL at a concentration of 1,000 μg / mL. E showed an activity of 91.8% and the RC50 value was measured at 43.5 μg / mL, indicating that the activity was similar to that of the radical scavenging activity of vitamin C (Table 2).

DPPH radical scavenging activity (%) of white tea extract sample Concentration (μg / mL) Rc ₅₀
10 100 1000 LPW (Hot Water Extraction) 13.7 ± 2.3 41.0 ± 3.2 75.2 ± 2.2 272.1 ± 1.9 LPA (High Pressure Extraction) 19.5 ± 1.9 41.4 ± 0.5 79.2 ± 0.8 242.1 ± 1.1 LPU (Ultrasound Extraction) 29.9 ± 1.1 38.1 ± 1.4 81.0 ± 1.0 281.5 ± 0.8 LPE (ethanol extraction) 19.2 ± 0.7 43.4 ± 0.5 82.3 ± 1.3 197.2 ± 2.4 Ascorbic acid 24.5 ± 0.5 47.5 ± 0.9 90.2 ± 0.8 135.3 ± 1.1

DPPH radical scavenging activity (%) of white acid tea fraction sample Concentration (μg / mL) Rc ₅₀
10 100 1000 Hexane fraction 22.7 ± 1.7 37.6 ± 2.1 83.2 ± 1.3 364.2 ± 1.3 Ethyl acetate fraction 48.0 ± 0.9 62.7 ± 1.1 91.8 ± 1.5 43.5 ± 0.9 Butanol fraction 36.2 ± 1.0 52.8 ± 1.7 80.6 ± 1.6 92.7 ± 0.9 Water fraction 20.2 ± 1.6 28.3 ± 2.3 68.7 ± 2.2 542.7 ± 1.4 Ascorbic acid 24.5 ± 0.5 47.5 ± 0.9 90.2 ± 0.8 135.3 ± 1.1

Example  3. ABTS Radical  Scavenging activity

DPPH dissolved in organic solvent has difficulty in measuring the antioxidant power of hydrophilic compounds. It has been reported that the maximum absorbance at can minimize the effects of pigments inherent in extracts and thus measure more accurate antioxidant power in DPPH. ABTS + radical cation scavenging activity is characterized by the reaction between ABTS and potassium persulfate. When ABTS + radical is produced, it becomes a distinctive index blue-green color. It can measure both hydrogen donating antioxidant and chain breaking antioxidant. In the ABTS radical scavenging activity, the activities of four extracts with different extraction methods of white acid tea were measured first, and the results were measured in the order of LPE> LPU> LPW> LPA at a concentration of 1,000 μg / mL, as shown in Table 3. LPE showed high ABTS radical scavenging activity of 99.8% at the highest concentration of 1,000 μg / mL. The ABTS radical scavenging activity of the solvent fraction of LPE with the highest activity was determined in the order of LPE / E> LPE / B> LPE / H> LPE / W at 1,000 μg / mL concentration. Also high scavenging activity of 99.6% was confirmed at a concentration of 1,000 μg / mL (Table 4). Based on these results, the white acid tea extract with the best radical scavenging activity was found to be LPE / E, a fraction of LPE and LPE, and their activity was similar to that of vitamin C even at concentrations of 10-100 μg / mL. The result is.

ABTS radical scavenging activity (%) of white tea extract sample Concentration (μg / mL) Rc ₅₀
10 100 1000 LPW (Hot Water Extraction) 12.4 ± 1.6 40.6 ± 4.1 92.1 ± 2.7 251.5 ± 0.8 LPA (High Pressure Extraction) 11.8 ± 2.7 39.2 ± 3.6 86.3 ± 1.3 273.9 ± 2.5 LPU (Ultrasound Extraction) 12.0 ± 0.9 45.7 ± 1.9 98.7 ± 2.4 158.5 ± 2.4 LPE (ethanol extraction) 9.4 ± 0.5 50.5 ± 2.5 99.8 ± 2.7 143.5 ± 1.7 Ascorbic acid 18.2 ± 0.9 49.7 ± 2.2 99.3 ± 1.7 156.5 ± 1.5

% ABTS radical scavenging activity of white acid fraction sample Concentration (μg / mL) Rc ₅₀
10 100 1000 Hexane fraction 10.2 ± 0.9 47.9 ± 4.1 97.5 ± 5.1 178.2 ± 4.2 Ethyl acetate fraction 26.1 ± 1.1 64.9 ± 3.2 99.6 ± 4.7 25.0 ± 2.2 Butanol fraction 16.8 ± 2.0 50.3 ± 1.9 98.3 ± 2.5 168.5 ± 2.9 Water fraction 7.8 ± 0.8 64.7 ± 1.2 80.7 ± 2.4 367.0 ± 1.5 Ascorbic acid 18.2 ± 0.9 49.7 ± 2.2 99.3 ± 1.7 156.5 ± 1.5

Example  4. Cytotoxicity Measurement

Toxicity in macrophage cells by the white tea extract and fractions is shown in Figure 1 by the MTT assay. The results of experiments to consider whether the anti-inflammatory effect of white tea extracts and fractions are due to the reduction of LPS-induced inflammation-related substances or the decrease of cell population due to the cytotoxicity of the extracts. And all four fractions showed a high survival of at least 90% at concentrations below 25 μg / mL. Therefore, NO, PGE2, TNF-α, IL-1β and IL-6 production inhibitory activity proceeded at the concentration below 25 μg / mL.

Example  5. NO (nitric oxide) production inhibitory activity

In inflammatory processes in the body, excess NO and inflammatory factors such as PGE2 (prostaglandin E2) are formed by inducible NO synthase (iNOS) and cycloxygenase (COX) -2. In general, the formation of NO plays an important role in killing bacteria or eliminating tumors, but excessive formation of NO by pathological causes causes inflammation, tissue damage, genetic variation and nerve damage. In order to examine whether the white tea extract has the inhibitory activity of NO production, first of all, the effects of NO production produced by LPS stimulation in Raw 264.7 cells were measured for the extracts according to four different white tea extract methods. All four extracts reduced NO production in cells in a concentration-dependent manner, but LPE showed 50% inhibitory activity at a concentration of 25 μg / mL. As a result of measuring the activity of the solvent fractions of LPE, the highest production inhibitory activity was confirmed in LPE / E, and the concentration of 100 μg / mL of the ethyl acetate fraction was shown to be 57% at 25 μg / mL. Showed better activity than that exhibited less than 50% inhibitory activity (FIG. 2).

Example  6. PGE2 (prostaglandin E2 A) production inhibitory activity

Prostaglandin is a locally activated substance and is an important factor that induces an inflammatory response and is synthesized by the action of cyclooxygenase (COX) from arachidonic acid. Among them, excessive PGE2 is expressed by the action of COX-2 (cycloxygenase-2) in the process of inflammation in the body, and PGE2 is a well-known inflammation-inducing factor that is involved in the movement of immune cells to the site of inflammation. Known. In this experiment, PGE2 production inhibitory activity was measured only for the LPE and LPE solvent fractions that showed the best results in the NO production inhibitory activity experiment. The effect of LPE and its solvent fractions on the inhibition of the production of excess PGE2, an inhibitor of inflammation in Raw 264.7 cells, was 70% inhibition at 25 μg / mL for LPE, and LPE / E for LPE / E. It also showed high inhibitory activity in a concentration-dependent manner. In particular, it showed a high inhibitory effect of 40% and 73% at concentrations of 12.5 and 25 μg / mL, respectively (FIG. 3). Therefore, it was confirmed that white tea ethanol extract and white tea ethyl acetate fraction strongly inhibited the production of PGE2, which showed a significant anti-inflammatory effect.

Example  7. Inflammatory cytokine production inhibitory activity

The inflammatory response involves various mediators such as pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6, prostagrandin and free radicals. In particular, IL-6 and IL-1β are exothermic and inflammatory mediators that stimulate the immune system and are known to be involved in tumor formation along with TNF-α. Materials that inhibit the overexpression of these inflammatory cytokines indicate the possibility of preventing or controlling various inflammatory diseases induced by the inflammatory response. In this study, the effects of LPE and its solvent fractions on the overexpression of TNF-α, IL-1β, and IL-6, which are inflammation regulators by LPS stimulation in Raw 264.7 cells, were measured. As a result, LPE and LPE / E concentration-dependently inhibited TNF-α, IL-1β, and IL-6 cytokines. LPE inhibited TNF-α, IL-1β, and IL-6 by 24%, 47%, and 40%, respectively, at concentrations of 25 μg / mL, resulting in better inhibitory activity in IL-1β than TNF-α and IL-6. Showed. In particular, LPE / E inhibited the production of TNF-α and IL-1β by 51% and 57%, respectively, at a concentration of 25 μg / mL and 62% of IL-6 at the same concentration (FIG. 4). ).

Claims (5)

Nitrogen oxide (NO), PGE2 (prostaglandin E2), TNF-α, IL-1β and IL-6, characterized in that it has an inhibitory effect, 70% ethanol 20 times (v) in Ledum palustre L. / w) Health functional food for antioxidant and anti-inflammatory, containing ethyl acetate fraction of baeksan tea ethanol extract extracted twice each 24 hours after the addition as an active ingredient. delete Nitrogen oxide (NO), PGE2 (prostaglandin E2), TNF-α, IL-1β and IL-6, characterized in that it has an inhibitory effect, 70% ethanol 20 times (v) in Ledum palustre L. / w) Anti-inflammatory and anti-inflammatory cosmetic composition containing ethyl acetate fraction of white acid tea ethanol extract extracted twice each 24 hours as an active ingredient. Nitrogen oxide (NO), PGE2 (prostaglandin E2), TNF-α, IL-1β and IL-6, characterized in that it has an inhibitory effect, 70% ethanol 20 times (v) in Ledum palustre L. / w) pharmaceutical composition for anti-inflammatory containing ethyl acetate fraction of white acid tea ethanol extract extracted twice each 24 hours as an active ingredient. delete

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